Human Prt1-like subunit protein (hPrt1) and human eIF4G-like protein (p97) genes

ABSTRACT

The present invention relates to novel human Prt1 (hPrt1) and eIF4G-like (p97) proteins which are involved in eukaryotic transcription In particular, isolated nucleic acid molecules are provided encoding the human hPrt1 and p97 proteins. hPrt1 and p97 polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of hPrt1 and p97 activity. Also provided are therapeutic methods for treating disease states associated with the hPrt1 and p97 proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of, and claims priority under 35U.S.C. § 120 to U.S. patent application No. 09/954,043, filed Sep. 18,2001, which is a divisional of, and claims priority under 35 U.S.C. §120 to U.S. patent application No. 09/546,238, filed Apr. 10, 2000, nowU.S. Pat. No. 6,316,225, which is a divisional of, and claims priorityunder 35 U.S.C. § 120 to U.S. patent application No. 08/990,140, filedDec. 12, 1997, now U.S. Pat. No. 6,093,795, which claims benefit under35 U.S.C. § 119(e) of U.S. Provisional Application No. 60/033,151, filedDec. 13, 1996. Each of the above referenced applications is herebyincorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to novel proteins involved in theinitiation of eukaryotic transcription. More specifically, isolatednucleic acid molecules are provided encoding a human Prt1-like subunitprotein (hPrt1) and a human eIF4G-like protein (p97). Also provided arehPrt1 and p97 polypeptides, as are vectors, host cells and recombinantmethods for producing the same. The invention further relates toscreening methods for identifying agonists and antagonists of hPrt1 andp97 activity.

[0004] 2. Related Art

[0005] Eukaryotic protein synthesis requires the participation oftranslation initiation factors, which assist in the binding of the mRNAto the 40S ribosomal subunit (reviewed in Merrick & Hershey, inTranslational Control, Hershey et al., eds., Cold Spring HarbourLaboratory Press, (1996), pp. 31-69 and Pain, Eur. J Biochem 236:747-771(1996)). Ribosome binding is facilitated by the cap structure (m⁷GpppN,where N is any nucleotide) that is present at the 5′ end of all cellularmRNAs (except organellar). Biochemical fractionation studies elucidatedthe general pathway for translation initiation and led to thecharacterization of several translation initiation factors (reviewed inMerrick & Hershey supra). It is believed that the mRNA cap structure isinitially bound by eukaryotic initiation factor (eIF) 4F, which, inconjunction with eIF4B, melts RNA secondary structure in the 5′untranslated region (UTR) of the mRNA to promote ribosome binding. eIF4Fis a more efficient RNA helicase than free eIF4A (Rozen et al., Mol.Cell. Biol. 10:1134-1144 (1990)), consistent with the idea that eIF4Arecycles through the eIF4F protein complex to function in unwinding(Pause et al., Nature 371:762-767 (1994)). The 40S ribosomal subunit, ina complex with eIF3, eIF1A and eIF2-GTP-tRNAimet, binds at or near thecap structure and scans vectorially the 5′ UTR in search of theinitiator AUG codon (reviewed in Merrick & Hershey, supra).

[0006] eIF3 is the largest translation initiation factor, with at least8 different polypeptide subunits and a total mass of approximately 550to 700 kDa (Schreier, et al., J Mol. Biol. 116:727-753 (1977); Benne &Hershey, Proc. Natl. Acacl. Sci. USA 73:3005-3009 (1976); Behlke et al.,Eur. J Biochem. 157:523-530 (1986)). In mammals, the apparent molecularmasses of the eIF3 subunits are 35, 36, 40, 44, 47, 66, 115 and 170 kDa(Behlke, supra; Meyer, et al., Biochemistry 21:4206-4212 (1982); Milburnet al., Arch. Biochem. Biophys 276:6-11 (1990)). eIF3 is a moderatelyabundant translation initiation factor, with 0.5 to 1 molecule perribosome in HeLa cells and rabbit reticulocyte lysates (Meyer, supra;Mengod & Trachsel, Biochem. Acta 825:169-174 (1985)). This proteincomplex assumes several functions during translation initiation(reviewed in Hannig, BioEssays 17:915-919 (1995)). eIF3 binds to the 40Sribosomal subunit and prevents joining with the 60S subunit. Itinteracts with the ternary complex and stabilizes the binding of thelatter to the 40S ribosomal subunit (Trachsel et al., J. Mol. Biol.116:755-767 (1977); Gupta et al., (1990); Goumans et al., Biochem.Biophys. Acta 608:39-46 (1980); Peterson et al., J. Biol. Chem.254:2509-2510 (1979)). eIF3 crosslinks to mRNA and 18S rRNA (Nygard &Westermann, Nucl. Acids Res. 10:1327-1334 (1982); Westermann & Nygard,Nucl. Acids Res. 12:8887-8897 (1984)), an activity mainly attributed tothe 66 kDa subunit (or 62 kDa in yeast; Garcia-Barrio, et al., GenesDev. 9:1781-1796 (1995); Naranda, et al., J. Biol. Chem. 269:32286-32292(1994)). eIF3 co-purifies with eIF4F and eIF4B, two initiation factorsinvolved in the mRNA binding step (Schreier et al., J. Mol. Biol.116:727-753 (1977)). A direct interaction between the 220 kDa subunit ofeIF4F and eIF3 has been demonstrated (Lamphear et al., J. Biol. Chem.270:21975-21983 (1995)) and a role for eIF3 serving as a bridge betweenthe 40S ribosomal subunit and eIF4F-bound mRNA has been postulated(Lamphear, supra).

[0007] The complex structure of eIF3 and its pleiotropic roles intranslation initiation have rendered the study of this factor difficult.The protein sequence for only three of the yeast subunits (SUI1/p16, p62and PRT1/p90) have been published (Garcia-Barrio et al., Genes Dev.9:1781-1796 (1995); Naranda, supra; Hanic-Joyce et al., J. Biol. Chem.262:2845-2851 (1987)). However, several other mammalian and yeastsubunits have been recently cloned. The yeast protein p90, also known asPrt1, is the most well characterized of those identified to date. Prt1is an integral subunit of eIF3 (Naranda, supra; Danaie et al., J. Biol.Chem. 270:4288-4292 (1995)). A conditional lethal mutation in the PRT1gene reduces the binding of the ternary to the 40S ribosomal subunit(Feinberg et al, J. Biol. Chem. 257:10846-10851 (1982)). Other mutationswhich confer temperature sensitivity are located in the central andcarboxy-terminal portion of Prt1. An N-terminal deletion which removesthe Prt1 putative RNA Recognition Motif (RRM; for reviews see Birney, etal., Nucl. Acids Res. 21:5803-5816 (1993); Burd & Dreyfuss, Science265:615-621 (1994b); Nagai et al., Trends Biochem. Sci. 20:235-240(1995)), acts a trans-dominant negative inhibitor (Evans et al., Mol.Cell. Biol. 15:4525-4535 (1995)).

[0008] Proteins that specifically inhibit cap-dependent translation havebeen described (Pause, supra; Lin et al., Science 266:653-656 (1994)):4E-binding protein-I and -2 (4E-BP1 and 4E-BP2) bind to eIF4E andprevent their association with eIF4G, because 4E-BPs and eIF4G share acommon site for eIF4E binding (Haghighat et al., EMBO J. 14:5701-5709(1995); Mader et al., Mol. Cell. Biol. 15:4990-4997 (1995)). Upontreatment of cells with insulin and growth factors, 4E-BPs becomephosphorylated. This leads to dissociation of the 4E-BPs from eIF4E andformation of the eIF4F complex, which results in stimulation oftranslation (Pause, supra; Lin, supra; Beretta, et al., EMBO J.15:658-664 (1996)).

SUMMARY OF THE INVENTION

[0009] The present invention provides isolated nucleic acid moleculescomprising polynucleotides encoding the hPrt1 and p97 polypeptideshaving the amino acid sequences shown in FIGS. 1A-1D (SEQ ID NO:2) andFIGS. 2A-2E (SEQ ID NO:4) or the amino acid sequences encoded by thecDNA clones deposited as ATCC Deposit Number 97766 on Oct. 18, 1996 andATCC Deposit Number 97767 on Oct. 18, 1996.

[0010] The present invention also relates to recombinant vectors, whichinclude the isolated nucleic acid molecules of the present invention,and to host cells containing the recombinant vectors, as well as tomethods of making such vectors and host cells and for using them forproduction of hPrt1 and p97 polypeptides or peptides by recombinanttechniques.

[0011] The invention further provides isolated hPrt1 and p97polypeptides having amino acid sequences encoded by polynucleotidesdescribed herein.

[0012] The present invention also provides a screening method foridentifying compounds capable of enhancing or inhibiting a cellularresponse induced by hPrt1 and/or p97 polypeptides, which involvescontacting cells which express hPrt1 and/or p97 polypeptides with thecandidate compound, assaying a cellular response, and comparing thecellular response to a standard cellular response, the standard beingassayed when contact is made in absence of the candidate compound;whereby, an increased cellular response over the standard indicates thatthe compound is an agonist and a decreased cellular response over thestandard indicates that the compound is an antagonist.

[0013] Additional aspects of the invention relate to methods fortreating an individual in need of either an increased or decreased levelof hPrt1 and/or p97 activity in the body comprising administering tosuch an individual a composition comprising a therapeutically effectiveamount of either an isolated hPrt1 and/or p97 polypeptides of theinvention (or an agonist thereof) or an hPrt1 and/or p97 antagonist.

[0014] The present invention also provides components for use in invitro translation systems. Two individual components of such translationsystems, hPrt1 and p97, are provided herein.

BRIEF DESCRIPTION OF THE FIGURES

[0015] FIGS. 1A-1D show the nucleotide (SEQ ID NO:1) and deduced aminoacid (SEQ ID NO:2) sequence of the hPrt1 polypeptide. The protein has amolecular weight of about 116 kDa, as shown in Example 1. The standardone-letter abbreviations for amino acids are used.

[0016] FIGS. 2A-2E show the nucleotide (SEQ ID NO:3) and deduced aminoacid (SEQ ID NO:4) sequence of the p97 polypeptide. The protein has amolecular weight of about 97 kDa, as shown in Example 2. Abbreviationsare as in FIGS. 1A-1D.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding an hPrt1 polypeptide having theamino acid sequence shown in FIGS. 1A-1D (SEQ ID NO:2). The hPrt1protein of the present invention shares sequence homology with the Prt1protein of Saccharomyces cerevisiae. The nucleotide sequence shown inFIGS. 1A-1D (SEQ ID NO:1) was obtained by sequencing a cDNA clone, whichwas deposited on Oct. 18, 1996 at the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110-2209, USA, and givenaccession number 97766. The deposited clone is contained in thepBluescript SK(−) plasmid (Stratagene, LaJolla, Calif.).

[0018] In addition, the present invention also provides isolated nucleicacid molecules comprising a polynucleotide encoding an p97 polypeptidehaving the amino acid sequence shown in FIGS. 2A-2E (SEQ ID NO:4). Thep97 protein of the present invention shares sequence homology with thehuman eIF4G protein. The nucleotide sequence shown in FIGS. 2A-2E (SEQID NO:3) was obtained by sequencing a cDNA clone, which was deposited onOct. 18, 1996 at the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209, USA, and given accession number97767. The deposited clone is contained in the pcDNAIII plasmid(Invitrogen, Inc.).

[0019] Nucleic Acid Molecules

[0020] Using the information provided herein, such as the nucleotidesequence in FIGS. 1A-1D (SEQ ID NO: 1), nucleic acid molecules of thepresent invention encoding the hPrt1 polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloningcDNAs using mRNA as starting material. While the hPRT1 gene was found tobe present in cDNA libraries produced from RNA from multiple tissues,the nucleic acid molecule described in FIGS. 1A-1D (SEQ ID NO: 1) wasisolated from a cDNA library derived from human bone marrow cells. Thedetermined nucleotide sequence of the hPrt1 cDNA of FIGS. 1A-1D (SEQ IDNO: 1) contains an open reading frame encoding a protein of 873 aminoacid residues, with an initiation codon at positions 97-99 of thenucleotide sequence in FIGS. 1A-1D (SEQ ID NO:1) and a molecular weightof about 116 kDa, as shown in Example 1. The hPrt1 protein shown inFIGS. 1A-1D (SEQ ID NO:2) is about 31% identical and about 50% similarto the Prt1 protein of Saccharomyces cerevisiae.

[0021] In addition, using the information provided herein, such as thenucleotide sequence in FIGS. 2A-2E (SEQ ID NO:3), a nucleic acidmolecule of the present invention encoding a p97 polypeptide may also beobtained using standard cloning and screening procedures. While the p97gene was identified in cDNA libraries produced from RNA from severaltissues, the nucleic acid molecule described in FIGS. 2A-2E (SEQ IDNO:3) was isolated from a cDNA library derived from human fetal heart.The determined nucleotide sequence of the p97 cDNA of FIGS. 2A-2E (SEQID NO:3) contains an open reading frame encoding a protein of 907 aminoacid residues, with an initiation codon at positions 307-309 of thenucleotide sequence in FIGS. 2A-2E (SEQ ID NO:3) and a molecular weightof about 97 kDa, as shown in Example 2. The p97 protein shown in FIGS.2A-2E (SEQ ID NO:4) is about 28% identical and about 36% similar toapproximately the C-terminal two thirds of eIF4G. The N-terminal thirdof eIF4G bears no similarity to the p97 protein of the presentinvention.

[0022] As one of ordinary skill would appreciate, due to thepossibilities of sequencing errors discussed above the actual hPrt1polypeptide encoded by the deposited cDNA comprises about 873 aminoacids, but may be anywhere in the range of about 850 to about 896 aminoacids. Similarly, the actual p97 polypeptide encoded by the depositedcDNA comprises about 907 amino acids, but may be anywhere in the rangeof about 882 to about 932 amino acids.

[0023] Nucleic acid molecules of the present invention may be in theform of RNA, such as mRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

[0024] By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintain ed in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules further includes such molecules produced synthetically.

[0025] Isolated nucleic acid molecules of the present invention includeDNA molecules comprising open reading frames (ORF) with initiationcodons at positions 97-99 of the nucleotide sequence shown in FIGS.1A-1D (SEQ ID NO:1) for hPrt1 and positions 307-309 of the nucleotidesequence shown in FIGS. 2A-2E (SEQ ID NO:3) for p97; and DNA moleculeswhich comprise a sequence substantially different from those describedabove but which, due to the degeneracy of the genetic code, still encodeeither the hPrt1 or p97 proteins.

[0026] In another aspect, the invention provides isolated nucleic acidmolecules encoding the hPrt1 and p97 polypeptides having amino acidsequences encoded by the cDNA clones contained in the plasmids depositedas ATCC Deposit No. 97766 on Oct. 18, 1996 and ATCC Deposit No. 97767 onOct. 18, 1996, respectively. The invention further provides isolatednucleic acid molecules having the nucleotide sequences shown in FIGS.1A-1D (SEQ ID NO:1), FIGS. 2A-2E (SEQ ID NO:3), the nucleotide sequenceof the hPrt1 and p97 cDNA contained in the above-described depositedclones, or a nucleic acid molecule having a sequence complementary toany one of the above sequences.

[0027] The present invention is further directed to fragments of theisolated nucleic acid molecules described herein. By a fragment of anisolated nucleic acid molecule having the nucleotide sequences of thedeposited cDNAs or the nucleotide sequences shown in FIGS. 1A-1D (SEQ IDNO:1) or FIGS. 2A-2E (SEQ ID NO:3) is intended fragments at least about15 nt, and more preferably at least about 20 nt, still more preferablyat least about 30 nt, and even more preferably, at least about 40 nt inlength which are useful as diagnostic probes and primers as discussedherein. Of course larger DNA fragments 50, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 100, 1050, 1100,1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700,1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300,2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900,2950, 3000, or 3010 nt in length of the sequence shown in SEQ ID NO:1are also useful according to the present invention as are fragmentscorresponding to most, if no all, of the nucleotide sequence of the cDNAclone contained in the plasmid deposited as ATCC Deposit No. 97766 or asshown in SEQ ID NO:1. Similarly, larger DNA fragments 50, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,100, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550,1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150,2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750,2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350,3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, or 3790 nt in length ofthe sequence shown in SEQ ID NO:3 are also useful according to thepresent invention as are fragments corresponding to most, if not all, ofthe nucleotide sequence of the cDNA clone contained in the plasmiddeposited by ATCC Deposit No. 97767 or as shown in SEQ ID NO:3. By afragment at least 20 nt in length, for example, is intended fragmentswhich include 20 or more contiguous bases from the nucleotide sequencesof the deposited cDNAs or the nucleotide sequences as shown in FIGS.1A-1D (SEQ ID NO:1) or FIGS. 2A-2E (SEQ ID NO:3). Since the genes havebeen deposited and the nucleotide sequences shown in FIGS. 1A-1D (SEQ IDNO:1) and FIGS. 2A-2E (SEQ ID NO:3) are provided, generating such DNAfragments would be routine to the skilled artisan.

[0028] Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding epitope-bearing portions of the hPrt1protein. In particular, such nucleic acid fragments of the presentinvention include nucleic acid molecules encoding: a polypeptidecomprising amino acid residues from about 1 to about 188 in FIGS. 1A-1D(SEQ ID NO:2); a polypeptide comprising amino acid residues from about193 to about 235 in FIGS. 1A-1D (SEQ ID NO:2); a polypeptide comprisingamino acid residues from about 248 to about 262 in FIGS. 1A-1D (SEQ IDNO:2); a polypeptide comprising amino acid residues from about 270 toabout 350 in FIGS. 1A-1D (SEQ ID NO:2); a polypeptide comprising aminoacid residues from about 361 to about 449 in FIGS. 1A-1D (SEQ ID NO:2);a polypeptide comprising amino acid residues from about 458 to about 620in FIGS. 1A-1D (SEQ ID NO:2); and a polypeptide comprising amino acidresidues from about 639 to about 846 in FIGS. 1A-1D (SEQ ID NO:2). Theinventors have determined that the above polypeptide fragments areantigenic regions of the hPrt1 protein. Methods for determining othersuch epitope-bearing portions of the hPrt1 protein are described indetail below.

[0029] Preferred nucleic acid fragments of the present invention alsoinclude nucleic acid molecules encoding epitope-bearing portions of thep97 protein. In particular, such nucleic acid fragments of the presentinvention include nucleic acid molecules encoding: a polypeptidecomprising amino acid residues from about 1 to about 98 in FIGS. 2A-2E(SEQ ID NO:4); a polypeptide comprising amino acid residues from about121 to about 207 in FIGS. 2A-2E (SEQ ID NO:4); a polypeptide comprisingamino acid residues from about 232 to about 278 in FIGS. 2A-2E (SEQ IDNO:4); a polypeptide comprising amino acid residues from about 287 toabout 338 in FIGS. 2A-2E (SEQ ID NO:4); a polypeptide comprising aminoacid residues from about 347 to about 578 in FIGS. 2A-2E (SEQ ID NO:4);a polypeptide comprising amino acid residues from about 593 to about 639in FIGS. 2A-2E (SEQ ID NO:4); a polypeptide comprising amino acidresidues from about 681 to about 770 in FIGS. 2A-2E (SEQ ID NO:4); apolypeptide comprising amino acid residues from about 782 to about 810in FIGS. 2A-2E (SEQ ID NO:4); and a polypeptide comprising amino acidresidues from about 873 to about 905 in FIGS. 2A-2E ( SEQ ID NO:4). Theinventors have determined that the above polypeptide fragments areantigenic regions of the p97 protein. Methods for determining other suchepitope-bearing portions of the p97 protein are also described in detailbelow.

[0030] In another aspect, the invention provides isolated nucleic acidmolecules comprising polynucleotides which hybridize under stringenthybridization conditions to a portion of a polynucleotide in nucleicacid molecules of the invention described above, for instances, the cDNAclones contained in ATCC Deposits Nos. 97766 and 97767. By “stringenthybridization conditions” is intended overnight incubation at 42° C. ina solution comprising: 50% formamide, 5×SSC (750mM NaCl, 75 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA,followed by washing the filters in ).1×SSC at about 65° C.

[0031] By polynucleotides which hybridize to a “portion” of apolynucleotide is intended a polynucleotide (either DNA or RNA)hybridizing to at least about 15 nucleotides (nt), and more preferablyat least about 20 nt, still more preferably at least about 30 nt, andeven more preferably about 30-70 nt of the reference polynucleotide.These are useful as diagnostic probes and primers as discussed above andin more detail below.

[0032] By a portion of a polynucleotide of “at least 20 nt in length,”for example, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide (e.g., the depositedcDNAs or the nucleotide sequences as shown in FIGS. 1A-1D (SEQ ID NO:1)and FIGS. 2A-2E (SEQ ID NO:3)). Of course, a polynucleotide whichhybridizes only to a poly A sequence (such as the 3 terminal poly(A)tract of the hPrt1 and p97 cDNAs, shown in FIGS. 1A-1D (SEQ ID NO:1) andFIGS. 2A-2E (SEQ ID NO:3)), or to a complementary stretch of T (or U)resides, would not be included in polynucleotides of the invention usedto hybridize to a portion of a nucleic acid of the invention, since sucha polynucleotide would hybridize to any nucleic acid molecule containinga poly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone).

[0033] Nucleic acid molecules of the present invention which encode thehPrt1 and p97 polypeptides may include, but are not limited to thoseencoding the amino acid sequences of the mature polypeptides, bythemselves; the coding sequences for the mature polypeptides andadditional sequences, such as those encoding amino acid leaders orsecretory sequences, such as a pre-, or pro- or prepro- proteinsequences; the coding sequences of the mature polypeptides, with orwithout the aforementioned additional coding sequences, together withadditional, non-coding sequences, including for example, but not limitedto introns and non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences that play a role in transcription, mRNAprocessing, including splicing and polyadenylation signals, forexample—ribosome binding and stability of mRNA; an additional codingsequence which codes for additional amino acids, such as those whichprovide additional functionalities. Thus, the sequences encoding thepolypeptides of the present invention may be fused to a marker sequence,such as a sequence encoding a peptide which facilitates purification ofthe fused polypeptide. In certain preferred embodiments of this aspectof the invention, the marker amino acid sequence is a hexa-histidinepeptide, such as the tag provided in a pQE vector (Qiagen, Inc.), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. The “HA” tag is another peptide useful for purification whichcorresponds to an epitope derived from the influenza hemagglutininprotein, which has been described by Wilson et al., Cell 37: 767 (1984).As discussed below, other such fusion proteins include the hPrt1 or p97fused to Fc at the N- or C-terminus.

[0034] The present invention further relates to variants of the nucleicacid molecules of the present invention, which encode portions, analogsor derivatives of the hPrt1 and p97 proteins. Variants may occurnaturally, such as a natural allelic variant. By an “allelic variant” isintended one of several alternate forms of a gene occupying a givenlocus on a chromosome of an organism. Genes II, Lewin, B., ed., JohnWiley & Sons, New York (1985). Non-naturally occurring variants may beproduced using art-known mutagenesis techniques.

[0035] Further embodiments of the invention include isolated nucleicacid molecules comprising a polynucleotide having a nucleotide sequenceat least 95% identical, and more preferably at least 96%, 97%, 98% or99% identical to (a) a nucleotide sequence encoding the full-lengthhPrt1 or p97 polypeptide having the complete amino acid sequence inFIGS. 1A-1D (SEQ ID NO:2) (amino acid residues 1 to 873) and FIGS. 2A-2E(SEQ ID NO:4) (amino acid residues 1 to 907); (b) a nucleotide sequenceencoding the full-length hPrt1 or p97 polypeptide having the completeamino acid sequence in FIGS. 1A-1D (SEQ ID NO:2) (amino acid residues 2to 873) and FIGS. 2A-2E (SEQ ID NO:4) (amino acid residues 2 to 907) butlacking the N-terminal amino acid residue; (c) a nucleotide sequenceencoding the hPrt1 or p97 polypeptide having the amino acid sequenceencoded by the cDNA clones contained in ATCC Deposit Nos. 97766 and97767, respectively; or (d) a nucleotide sequence complementary to anyof the nucleotide sequences in (a), (b) or (c).

[0036] By a polynucleotide having a nucleotide sequence at least, forexample, 95% “identical” to a reference nucleotide sequence encodingeither an hPrt1 or p97 polypeptide is intended that the nucleotidesequence of a polynucleotide which is identical to the referencesequence except that the polynucleotide sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequences encoding the hPrt1 or p97 polypeptides. In other words, toobtain a polynucleotide having a nucleotide sequence at least 95%identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. These mutations of the reference sequence may occur at the 5or 3 terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence.

[0037] As a practical matter, whether any particular nucleic acidmolecule is at least 95%, 96%, 97%, 98% or 99% identical to, forinstance, the nucleotide sequences shown in FIGS. 1A-1D (SEQ ID NO:1)and FIGS. 2A-2E (SEQ ID NO:3) or to the nucleotide sequences of thedeposited cDNA clones can be deternined conventionally using knowncomputer programs such as the Bestfit program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).Bestfit uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2: 482-489 (1981)), to find the bestsegment of homology between two sequences. When using Bestfit or anyother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed.

[0038] The present application is directed to nucleic acid molecules atleast 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequencesshown in FIGS. 1A-1D (SEQ ID NO: 1), FIGS. 2A-2E (SEQ ID NO:3), or tothe nucleic acid sequence of the deposited cDNAs, irrespective ofwhether they encode a polypeptide having hPrt1 or p97 activity. This isbecause even where a particular nucleic acid molecule does not encode apolypeptide having such activity, one of skill in the art would stillknow how to use the nucleic acid molecule, for instance, as ahybridization probe or a polymerase chain reaction (PCR) primer.

[0039] Preferred, however, are nucleic acid molecules having sequencesat least 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequences shown in FIGS. 1A-1D (SEQ ID NO:1), FIGS. 2A-2E (SEQ ID NO:3),or to the nucleic acid sequences of the deposited cDNAs which do, infact, encode a polypeptide having hPrt1 or p97 protein activity. By “apolypeptide having hPrt1 or p97 activity” is intended polypeptidesexhibiting activity similar, but not necessarily identical, to anactivity of either the hPrt1 or p97 protein of the invention, asmeasured in a particular biological assay. For instance, p97 proteinactivity can be measured using the ability of a p97 homolog to eithersuppress translation or bind to eIF4A or eIF3, as described in theExamples below. hPrt1 protein activity can be measured, for example,using the ability of an hPrt1 homolog to interact with proteins in theeIF3 complex, also as described in the Examples below.

[0040] Of course, due to the degeneracy of the genetic code, one ofordinary skill in the art will immediately recognize that a large numberof the nucleic acid molecules having a sequence at least 95%, 96%, 97%,98%, or 99% identical to the nucleic acid sequences of the depositedcDNAs or the nucleic acid sequences shown in FIGS. 1A-1D (SEQ ID NO:1)or FIGS. 2A-2E (SEQ ID NO:3) will encode a polypeptide “having hPrt1 orp97 protein activity.” In fact, since degenerate variants of thesenucleotide sequences all encode the same polypeptide, this will be clearto the skilled artisan even without performing the above describedcomparison assay. It will be further recognized in the art that, forsuch nucleic acid molecules that are not degenerate variants, areasonable number will also encode polypeptides having hPrt1 or p97protein activity.

[0041] For example, guidance concerning how to make phenotypicallysilent amino acid substitutions is provided in Bowie, J. U. et al.,“Deciphering the Message in Protein Sequences: Tolerance to Amino AcidSubstitutions,” Science 247:1306-1310 (1990), wherein the authorsindicate that there are two main approaches for studying the toleranceof an amino acid sequence to change. The first method relies on theprocess of evolution, in which mutations are either accepted or rejectedby natural selection. The second approach uses genetic engineering tointroduce amino acid changes at specific positions of a cloned gene andselections or screens to identify sequences that maintain functionality.These studies have revealed that proteins are surprisingly tolerant ofamino acid substitutions. The authors further indicate which amino acidchanges are likely to be permissive at a certain position of theprotein. Numerous phenotypically silent substitutions are described inBowie, J. U. et al., supra, and the references cited therein.

[0042] Vectors and Host Cells

[0043] The present invention also relates to vectors which include theisolated DNA molecules of the present invention, host cells which aregenetically engineered with the recombinant vectors, and the productionof hPrt1 and p97 polypeptides or fragments thereof by recombinanttechniques.

[0044] The polynucleotides may be joined to a vector containing aselectable marker for propagation in a host. The DNA insert should beoperatively linked to an appropriate promoter, such as the phage lambdaPL promoter, the E. coli lac, trp and tac promoters, the SV40 early andlate promoters and promoters of retroviral LTRs, to name a few. Othersuitable promoters will be known to the skilled artisan. The expressionconstructs will further contain sites for transcription initiation,termination and, in the transcribed region, a ribosome binding site fortranslation. The coding portion of the mature transcripts expressed bythe constructs will preferably include a translation initiating at thebeginning and a termination codon (UAA, UGA or UAG) appropriatelypositioned at the end of the polypeptide to be translated.

[0045] As indicated, the expression vectors will preferably include atleast one selectable marker. Such markers include dihydrofolatereductase or neomycin resistance for eukaryotic cell culture andtetracycline or ampicillin resistance genes for culturing in E. coli andother bacteria. Representative examples of appropriate hosts include,but are not limited to, bacterial cells, such as E. coli, Streptomycesand Salmonella typhimurium cells; fungal cells, such as yeast cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS and Bowes melanoma cells; and plant cells.

[0046] Among vectors preferred for use in bacteria include pQE70, pQE60and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available fromStratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG andpSVL available from Pharmacia.

[0047] Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

[0048] The polypeptide may be expressed in a modified form, such as afusion protein, and may include not only secretion signals, but alsoadditional heterologous functional regions. For instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification, or during subsequenthandling and storage. Also, peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. A preferred fusionprotein comprises a heterologous region from immunoglobulin that isuseful to solubilize proteins. For example, EP-A-O 464 533 (Canadiancounterpart 2045869) discloses fusion proteins comprising variousportions of constant region of immunoglobin molecules together withanother human protein or part thereof. In many cases, the Fe part in afusion protein is thoroughly advantageous for use in therapy anddiagnosis and thus results, for example, in improved pharmacokineticproperties (EP-A 0232 262). On the other hand, for some uses it would bedesirable to be able to delete the Fe part after the fusion protein hasbeen expressed, detected and purified in the advantageous mannerdescribed. This is the case when Fe portion proves to be a hindrance touse in therapy and diagnosis, for example when the fusion protein is tobe used as antigen for immunizations. In drug discovery, for example,human proteins, such as, hIL-5 receptor has been fused with Fe portionsfor the purpose of high-throughput screening assays to identifyantagonists of hIL-5. See, D. Bennett et al., Journal of MolecularRecognition, Vol. 8:52-58 (1995) and K. Johanson et al., The Journal ofBiological Chemistry, Vol. 270, No. 16:9459-9471 (1995).

[0049] The hPrt1 and p97 proteins can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated.

[0050] hPrt1 and p97 Polypeptides and Fragments

[0051] The invention further provides isolated hPrt1 and p97polypeptides having the amino acid sequences encoded by the depositedcDNAs, or the amino acid sequences shown in FIGS. 1A-1D (SEQ ID NO:2)and FIGS. 2A-2E (SEQ ID NO:4), or a peptide or polypeptide comprising aportion of the above polypeptides.

[0052] It will be recognized in the art that some amino acid sequencesof the hPrt1 and p97 polypeptides can be varied without significanteffect on the structure or function of the proteins. Thus, the inventionfurther includes variations of the hPrt1 and p97 polypeptides which showsubstantial hPrt1 and p97 polypeptide activities or which includeregions of hPrt1 and p97 proteins such as the portions discussed below.Such mutants include deletions, insertions, inversions, repeats, andtype substitutions. As indicated above, guidance concerning which aminoacid changes are likely to be phenotypically silent can be found inBowie, J. U., et al., “Deciphering the Message in Protein Sequences:Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990).

[0053] Thus, the fragment, derivative or analog of the polypeptides ofFIGS. 1A-1D (SEQ ID NO:2), FIGS. 2A-2E (SEQ ID NO:4), or those encodedby the deposited cDNAs, may be (i) one in which one or more of the aminoacid residues are substituted with a conserved or non-conserved aminoacid residue (preferably a conserved amino acid residue) and suchsubstituted amino acid residue may or may not be one encoded by thegenetic code, or (ii) one in which one or more of the amino acidresidues includes a substituent group, or (iii) one in which thepolypeptide is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol), or (iv) one in which the additional amino acids are fused tothe polypeptide.

[0054] Of particular interest are substitutions of charged amino acidswith another charged amino acid and with neutral or negatively chargedamino acids. The latter results in proteins with reduced positive chargeto improve the characteristics of the hPrt1 and p97 proteins. Theprevention of aggregation is highly desirable. Aggregation of proteinsnot only results in a loss of activity but can also be problematic whenpreparing pharmaceutical formulations, because they can be immunogenic.(Pinckard et al., Clin Exp. Immunol. 2:331-340 (1967); Robbins et al.,Diabetes 36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic DrugCarrier Systems 10:307-377 (1993)).

[0055] As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table 1). TABLE 1Conservative Amino Acid Substitutions. Aromatic Phenylalanine TryptophanTyrosine Hydrophobic Leucine Isoleucine Valine Polar GlutamineAsparagine Basic Arginine Lysine Histidine Acidic Aspartic Acid GlutamicAcid Small Alanine Serine Threonine Methionine Glycine

[0056] Amino acids in the hPrt1 and p97 proteins of the presentinvention that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). Thelatter procedure introduces single alanine mutations at every residue inthe molecule. The resulting mutant molecules are then tested forbiological activity such the ability to bind to cellular transcriptionfactors or RNA.

[0057] The polypeptides of the present invention are preferably providedin an isolated form. By “isolated polypeptide” is intended a polypeptideremoved from its native environment. Thus, a polypeptide produced andcontained within a recombinant host cell would be considered “isolated”for purposes of the present invention. Also intended as an “isolatedpolypeptide” are polypeptides that have been purified, partially orsubstantially, from a recombinant host. For example, recombinantlyproduced versions of the hPrt1 and p97 polypeptides can be substantiallypurified by the one-step method described in Smith and Johnson, Gene67:31-40 (1988).

[0058] The polypeptides of the present invention include thepolypeptides encoded by the deposited cDNAs; a polypeptide comprisingamino acids about I to about 873 in SEQ ID NO:2; a polypeptidecomprising amino acids about 1 to about 907 in SEQ ID NO:4; apolypeptide comprising amino acids about 2 to about 873 in SEQ ID NO:2;a polypeptide comprising amino acids about 2 to about 907 in SEQ IDNO:4; as well as polypeptides which are at least 90% or 95% identical,more preferably at least 96% identical, still more preferably at least97%, 98% or 99% identical to those described above and also includeportions of such polypeptides with at least 30 amino acids and morepreferably at least 50 amino acids.

[0059] The polypeptides of the present invention include polypeptides atleast 90% or 95% identical, more preferably at least 96% identical, andstill more preferably at least 97%, 98% or 99% identical to either thepolypeptides encoded by the deposited cDNAs or the polypeptides of FIG.1A-1D (SEQ ID NO:2) or FIG. 2A-2E (SEQ ID NO:4), and also includeportions of such polypeptides with at least 30 amino acids and morepreferably at least 50 amino acids.

[0060] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a reference amino acid sequence of an hPrt1or p97 polypeptide is intended that the amino acid sequence of thepolypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of either the hPrt1 orp97 polypeptide. In other words, to obtain a polypeptide having an aminoacid sequence at least 95% identical to a reference amino acid sequence,up to 5% of the amino acid residues in the reference sequence may bedeleted or substituted with another amino acid, or a number of aminoacids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence.

[0061] As a practical matter, whether any particular polypeptide is atleast 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequence shown in FIGS. 1A-1D (SEQ ID NO:2), FIGS. 2A-2E (SEQ IDNO:4) or to the amino acid sequence encoded by one of the deposited cDNAclones can be determined conventionally using known computer programssuch the Bestfit program (Wisconsin Sequence Analysis Package, Version 8for Unix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). When using Bestfit or any other sequencealignment program to determine whether a particular sequence is, forinstance, 95% identical to a reference sequence according to the presentinvention, the parameters are set, of course, such that the percentageof identity is calculated over the full length of the reference aminoacid sequence and that gaps in homology of up to 5% of the total numberof amino acid residues in the reference sequence are allowed.

[0062] The polypeptides of the present invention is useful as amolecular weight marker on SDS-PAGE gels or on molecular sieve gelfiltration columns using methods well known to those of skill in theart.

[0063] In another aspect, the invention provides a peptide orpolypeptide comprising an epitope-bearing portion of the polypeptides ofthe invention. The epitope of these polypeptide portions is animmunogenic or antigenic epitope of polypeptides described herein. An“immunogenic epitope” is defined as a part of a protein that elicits anantibody response when the whole protein is the immunogen. On the otherhand, a region of a protein molecule to which an antibody can bind isdefined as an “antigenic epitope.” The number of immunogenic epitopes ofa protein generally is less than the number of antigenic epitopes. See,for instance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002(1983).

[0064] It is well known in that art that relatively short syntheticpeptides that mimic part of a protein sequence are routinely capable ofeliciting an antiserum that reacts with the partially mimicked protein.See, for instance, Sutcliffe, J. G., Shinnick, T. M., Green, N. andLearner, R., Science 219:660-666 (1983). Peptides capable of elicitingprotein-reactive sera are frequently represented in the primary sequenceof a protein, can be characterized by a set of simple chemical rules,and are confined neither to immunodominant regions of intact proteins(i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.

[0065] Antigenic epitope-bearing peptides and polypeptides of theinvention are therefore useful to raise antibodies, including monoclonalantibodies, that bind specifically to polypeptides of the invention.See, for instance, Wilson et al., Cell 37:767-778 (1984) at 777.

[0066] Antigenic epitope-bearing peptides and polypeptides of theinvention preferably contain a sequence of at least seven, morepreferably at least nine and most preferably between about at leastabout 15 to about 30 amino acids contained within the amino acidsequence of polypeptides of the invention.

[0067] Non-limiting examples of antigenic polypeptides or peptides thatcan be used to generate hPrt1-specific antibodies include: a polypeptidecomprising amino acid residues from about 1 to about 188 in FIGS. 1A-1D(SEQ ID NO:2); a polypeptide comprising amino acid residues from about193 to about 235 in FIGS. 1A-1D (SEQ ID NO:2); a polypeptide comprisingamino acid residues from about 248 to about 262 in FIGS. 1A-1D (SEQ IDNO:2); a polypeptide comprising amino acid residues from about 270 toabout 350 in FIGS. 1A-1D (SEQ If) NO:2); a polypeptide comprising aminoacid residues from about 361 to about 449 in FIGS. 1A-1D (SEQ ID NO:2);a polypeptide comprising amino acid residues from about 458 to about 620in FIGS. 1A-1D (SEQ ID NO:2); and a polypeptide comprising amino acidresidues from about 639 to about 846 in FIGS. 1A-1D (SEQ ID NO:2). Asindicated above, the inventors have determined that the abovepolypeptide fragments are antigenic regions of the hPrt1 protein.

[0068] In addition, non-limiting examples of antigenic polypeptides orpeptides that can be used to generate p97-specific antibodies include: apolypeptide comprising amino acid residues from about 1 to about 98 inFIGS. 2A-2E (SEQ ID NO:4); a polypeptide comprising amino acid residuesfrom about 121 to about 207 in FIGS. 2A-2E (SEQ ID NO:4); a polypeptidecomprising amino acid residues from about 232 to about 278 in FIGS.2A-2E (SEQ ID NO:4); a polypeptide comprising amino acid residues fromabout 287 to about 338 in FIGS. 2A-2E (SEQ ID NO:4); a polypeptidecomprising amino acid residues from about 347 to about 578 in FIGS.2A-2E (SEQ ID NO:4); a polypeptide comprising amino acid residues fromabout 593 to about 639 in FIGS. 2A-2E (SEQ ID NO:4); a polypeptidecomprising amino acid residues from about 681 to about 770 in FIGS.2A-2E (SEQ ID NO:4); a polypeptide comprising amino acid residues fromabout 782 to about 810 in FIGS. 2A-2E (SEQ ID NO:4); and a polypeptidecomprising amino acid residues from about 873 to about 905 in FIGS.2A-2E (SEQ ID NO:4). As indicated above, the inventors have determinedthat the above polypeptide fragments are antigenic regions of the p97protein.

[0069] The epitope-bearing peptides and polypeptides of the inventionmay be produced by any conventional means. Houghten, R. A., Proc. Natl.Acad. Sci. USA 82:5131-5135 (1985). This “Simultaneous Multiple PeptideSynthesis (SMPS)” process is further described in U.S. Pat. No.4,631,211.

[0070] As one of skill in the art will appreciate, the hPrt1 and p97polypeptides of the present invention and the epitope-bearing fragmentsthereof described above can be combined with parts of the constantdomain of immunoglobulins (IgG), resulting in chimeric polypeptides.Fusion proteins that have a disulfide-linked dimeric structure due tothe IgG part can be more efficient in binding and neutralizing othermolecules than the monomeric protein or protein fragment alone(Fountoulakis et al., J Biochem 270:3958-3964 (1995)).

[0071] hPrt1 and p97 Polypeptides: Use for Screening for Agonists andAntagonists of hPrt1 and p97 Polypeptide Function

[0072] In one aspect, the present invention is directed to a method forenhancing an activity of an hPrt1 (e.g., modulation of apoptosis, theability to bind RNA or other known cellular ligands (such as p170 andeIF4G), participation in the process of translation) or p97 (e.g.,modulation of apoptosis, the ability to bind eIF4A and eIF3, suppressionof translation) polypeptide of the present invention, which involvesadministering to a cell which expresses the hPrt1 and/or p97 polypeptidean effective amount of an agonist capable of increasing an activity ofeither the hPrt1 or p97 protein. Preferably, the hPrt1 or p97polypeptide mediated activity is increased to treat a disease.

[0073] In a further aspect, the present invention is directed to amethod for inhibiting an activity of an hPrt1 or p97 polypeptide of thepresent invention, which involves administering to a cell whichexpresses the hPrt1 and/or p97 polypeptide an effective amount of anantagonist capable of decreasing an activity of either the hPrt1 or p97protein. Preferably, hPrt1 or p97 polypeptide mediated activity isdecreased to also treat a disease.

[0074] By “agonist” is intended naturally occurring and syntheticcompounds capable of enhancing or potentiating an activity of an hPrt1or p97 polypeptide of the present invention. By “antagonist” is intendednaturally occurring and synthetic compounds capable of inhibiting anactivity of an hPrt1 or p97 polypeptide. Whether any candidate “agonist”or “antagonist” of the present invention can enhance or inhibit anactivity can be determined using art-known assays, including thosedescribed in more detail below.

[0075] Thus, in a further aspect, a screening method is provided fordetermining whether a candidate agonist or antagonist is capable ofenhancing or inhibiting a cellular activity of an hPrt1 or p97polypeptide. The method involves contacting cells which express one orboth of the hPrt1 or p97 polypeptides with a candidate compound,assaying a cellular response, and comparing the cellular response to astandard cellular response, the standard being assayed when contact ismade with the polypeptide(s) in absence of the candidate compound,whereby an increased cellular response over the standard indicates thatthe candidate compound is an agonist of the polypeptide activity and adecreased cellular response compared to the standard indicates that thecandidate compound is an antagonist of the activity. By “assaying acellular response” is intended qualitatively or quantitatively measuringa cellular response to a candidate compound and either an hPrt1 or p97polypeptide (e.g., modulation of apoptosis, the ability to bind RNA orother known cellular ligands, participation in the process oftranslation).

[0076] Potential antagonists include the hPrt1 RRM and fragmentsthereof, e.g., hPrt1 polypeptide fragments that include the RNA bindingdomain. Such forms of the protein, which may be naturally occurring orsynthetic, antagonize hPrt1 polypeptide mediated activity by competingfor binding to RNA. Thus, such antagonists include fragments of thehPrt1 that contain the ligand binding domains of the polypeptides of thepresent invention.

[0077] Additional agonists according to the present invention includefragments of the p97 polypeptide capable of suppressing translation.

[0078] Proteins and other compounds which bind the hPrt1 or p97polypeptide domains are also candidate agonist and antagonist accordingto the present invention. Such binding compounds can be “captured” usingthe yeast two-hybrid system (Fields and Song, Nature 340:245-246 (1989);Gyuris et al., Cell 75:791-803 (1993); Zervos et al., Cell 72:223 -232(1993)).

[0079] hPrt1 and p97 polypeptide antagonists also include smallmolecules which bind to and occupies active regions of the hPrt1 or p97polypeptide thereby making the polypeptide inaccessible to ligands whichbind thereto such that normal biological activity is prevented. Examplesof small molecules include but are not limited to nucleotide sequencesand small peptides or peptide-like molecules. Such molecules may beproduced and screened for activity by a variety of methods (e.g., Lightand Lerner, Bioorganic & Medicinal Chemistry 3(7):955-967 (1995); Chenget al., Gene 171:1-8 (1996); Gates et al., J. Mol. Biol. 255:373-386(1996)).

[0080] In Vitro Translation Systems

[0081] The polypeptides of the present invention are also valuable foruse in in vitro translation systems. The events leading to theinitiation of protein synthesis in eukaryotic cells have been studiedusing both reconstitution of translational systems using purifiedcomponents and, more recently, genetic analyses. Hannig, BioEssays17(11):915-919 (1995). There remains, however, a current need foridentifying molecules involved in translation and the role each of thosemolecules play in the process of translation. The present inventionprovides two such molecules: hPrt1 and p97.

[0082] Several commercially available kits are currently on the marketfor performing translation in vitro. See, e.g., Boehringer Mannheim,Indianapolis, Ind., Cat. Nos:1559 451, 1103 059, 1103 067; LifeTechnologies, Grand Island, N.Y., Cat. Nos:18127-019, 18128-017. Thesekits generally provide lysates derived from either whole animal or plantcells which are capable of producing protein from mRNA. These lysatesare generally used as part of a translation reaction mixture whichcontains, in addition to the lysate, mRNA and both labeled and unlabeledamino acids. Thus, while the process of translation can generally beperformed to produce a protein of interest, the mechanism by which thoseproteins are produced has not yet been fully elucidated.

[0083] The present invention provides individual components of thesecell lysates which are useful for studying the process of translation.The p97 protein, for example, as a putative competitive inhibitor ofeIF4G which suppresses both cap dependent and independent translation,is useful for identifying mechanisms by which gene expression isregulated at the translational level. The p97 protein may also be usefulfor identifying specific genes which are regulated at the translationallevel.

[0084] Similarly, the present invention also provides the hPrt1 proteinwhich is believed to be both a member of the eIF3 complex and anecessary component of translation systems. In order for researchers tofully reconstitute a translation system from individual proteins each ofthe proteins of that system must be identified and available in purifiedform. The present invention provides the hPrt1 protein as one of thosecomponents. Such reconstitution studies will be useful in elucidatingthe specific role of each component of the system. For example, theprocesses of both initiation of proteins synthesis and elongation of theresulting polypeptide chain can be studied by either altering the ratiosof the various components or leaving one or more component out of thereaction mixture.

[0085] In addition, the present invention provides fragments andhomologs of the hPrt1 and p97 polypeptides, produced as described above,which act as either agonists or antagonists of the hPrt1 and p97proteins. Such fragments of the hPrt1 polypeptide may be useful, forexample, for inhibiting translation by blocking the binding of nativehPrt1 with either other proteins to form the eIF3 complex or RNA. Inaddition, such fragments of the p97 polypeptide may be useful, forexample, for competitively inhibiting eIF4G.

[0086] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

EXAMPLE 1

[0087] Materials

[0088] Materials were obtained from the following sources: T7 DNApolymerase sequencing kit, Pharmacia LKB Biotechnologies. ProteinA-Sepharose, Repligen. Heart muscle kinase, Sigma. Hybond-N+nylonmembrane, chemiluminescence system, Amersham. Poyvinylidene fluoridemembrane, Millipore. ³²P-ATP (6000 Ci/mmol), α³²P dCTP (3000 Ci/mmol),³⁵S-methionine (1000 Ci/mmol), DuPont-NEN. Oligonucleotides wereprepared at the Sheldon Biotechnology Centre, McGill University, Canada.

[0089] Isolation of hPrt1 cDNA clones

[0090] The expressed sequence tag used in this study (EST #112738 fromHuman Genome Science (HGS) Inc.) was identified using established ESTmethods described previously (Adams, M. D., et al., Nature (London)377:3-174 (1995)), and this partial cDNA clone encoding the humanhomologue of the yeast Prt1 protein was used to obtain the full lengthcDNA clone. Full length cDNA clones for hPrt1 were isolated from a gt11human placenta cDNA library. A 250 bp DNA was generated by thepolymerase chain reaction (PCR) using the hPrt1 EST clone as template.The amplified DNA was 32p labeled by random priming using a ³²p dCTP,random hexamers and the Klenow fragment of DNA polymerase (Feinberg, A.P., & Vogelstein, B., Analytical Biochem. 137:266-267 (1984)), and usedas a probe in CDNA screening and Northern blot analysis. For cDNAscreening, 5×105 phages displayed on duplicate sets of filters(Hybond-N+, Amersham) were prehybridized in 5X SSPE (20X SSPE is 3.6 MNaCl, 0.2 M Na P04, 0.02 M EDTA, pH 7.7), 5X Denhardt's solution (IXDenhardt's is 0.1% bovine serum albumin, 0.1% Ficoll, 0.1%polyvinylpyrrolidone), 9.5% SDS and 40 μg/ml heat-denatured salmon spermDNA, for 4 hours at 65° C. Hybridization was performed in the samebuffer containing the hPrt1 probe at 1×10⁶ cpm/ml for 16 hours at 65° C.Filters were washed to a final stringency of 0.1X SSPE/0.1% SDS at 65°C., and exposed to Kodak XAR films for 72 hours with intensifyingscreens. Phages from positive clones were used to prepare plate lysatesand DNA was purified, digested with SalI and ligated into pBluescriptthat had been digested with SalI. Oligonucleotides used for sequencingwere derived from either pBluescript or from the hPrt1 EST DNA sequence.The nueleotide sequence for full length hPrt1-1 was obtained from bothstrands of independent overlapping clones using the dideoxy chaintermination method (Sanger, F., et al., Proc. Natl. Acad. Sci. USA74:5463-5467 (1977)) and the T7 polymerase sequencing kit (Pharmacia).Regions of compression were re-sequenced using 7-deaza dGTP.

[0091] Vectors, Proteins

[0092] The full length cDNA (clone 3-6) was excised from the gt11 phageby SalI digestion and inserted into pBluescript KS in the T7 promoterorientation. The resulting vector is designated as KST7hPrt1-6.Constructs for truncated hPrt1 proteins were generated by PCR usingprimers in which an EcoRI site had been engineered. Cleavage of the PCRproduct with EcoRI and ligation into pAR90[59/69] (Blanar, M. A., &Rutter, W. J., Science 256:1014-1018 (1992)) or pGEX2T[128/129] (Blanar,M. A., & Rutter, W, supra) that had been digested with EcoRI preservesthe hPrt1 open reading frame and creates a GST-FLAG-HMK or FLAG-HMKfusion protein. For pAR90 N90146, the forward (5′) primer was 5′ACCGGAATTCAAAATGGACGCGGACGAGCCCTC 3′ (SEQ ID NO:5) and the reverse (3′)was primer 5′ AGCGGAATTCTTAAATCCCCCACTGCAG 3′ (SEQ ID NO:6). For pGEXN255, the hPrt1 open reading frame was first amplified by PCR andinserted into pGEX2T[128/129]. The resulting vector was linearized withHindIII, blunt ended with the Klenow fragment of E. coli DNA polymeraseand religated. Religation creates a stop codon 3 amino acids downstreamof hPrt1 residue 255. pGEX 146-255 was obtained by linearizing pGEXN90146 with HindIII, blunt-ending with Klenow and religating. Vectorswere transformed in either E. coli BL21 or BL21 pLysS. Bacteria weregrown in LB broth to an optical density of 0.5 and protein expressionwas induced with 1 mM IPTG (isopropyl-b-D-thiogalactopyranoside) for 1 hat 37° C. Cells were pelleted and lysed in lysis buffer (PBS, 1 mM EDTA,1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride) by 6 sonication cycles.Debris was removed by centrifugation. GST fusion proteins were purifiedon glutathione-Sepharose (Pharmacia) as described previously (Methot,N., et al., Molecular and Cell Biology 14:2307-2316 (1994)). FLAG-HMKfusion proteins were affinity-purified over an anti-FLAG column (Kodak)according to the manufacturer's specifications. pACTAG-hPrt1 was made bylinearizing pACTAG-2 (Charest, A., et al., Biochem. J. 308:425-432(1995)) with NotI, and inserting the hPrt1 ORF that had been excisedfrom KST7hPrt1 cut with NotI. hPrt1 is expressed from this vector as afusion protein bearing three hemagglutinin (HA) tags at its aminoterminus.

[0093] In Vitro Transcription and Translation

[0094] KST7hPrt1-6 was digested with DraI, and the linearized plasmidwas used as template for in vitro transcription using T7 RNA polymerase(Promega) under conditions recommended by the supplier. Translationreactions were performed in nuclease-treated rabbit reticulocyte lysate(Promega) in a final volume of 15 μl. Reaction mixtures contained 10 μllysate, 10 μCi ³⁵S-methionine (1000 Ci/Mmole), 15 U RNAsin (Promega), 20μM amino acid mixture (minus methionine) and 100 ng RNA. The reactionswere incubated 60 minutes at 30° C. and stopped by the addition of 3volumes of Laemmli buffer. Translation products were analyzed by SDS-9%polyacrylamide gel electrophoresis. Gels were fixed, treated in 16%salicylic acid, dried and processed for autoradiography.

[0095] Immunoprecipitations, Western Blots of hPrt1 Polypeptides

[0096] For HA-hPrt1 protein expression, HeLa cells that had beencultured in Dubelco DMEM media supplemented with 10% fetal bovine serum(FBS) were infected for 1 hour with recombinant vaccinia virus vTF7-3with the T7 RNA polymerase cDNA inserted into its genome (Fuerst, T. R.,et al., Proc. Natl. Acad. Sci. USA 83:8122-8126 (1986)), and transfectedwith 5 μg plasmid DNA using lipofectine (Invitrogen Life Technologies,Carlsbad, Calif.) in DMEM without FBS. Cells were incubated 2 hours withthe DNA-lipofectine mixture, and returned to DMEM-10% FBS for 12 hoursbefore harvesting. The cells were lysed in 20 mM Tris-HCl pH 7.4, 75 mMKCl, 5 mM MgCl , 1 mM DTT, 10 % glycerol, 1% Triton X-100, 1 mnM PMSF,aprotinin (25 ng/ml) and pepstatin (1 ng/ml). Cellular debris and nucleiwere removed by centrifugation, and protein content was assayed by theBradford method. Immunoprecipitations were performed on 200 μg ofextract using a-HA antibody. Briefly, extracts were diluted to 500 μl inRIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5%Na-deoxycholate) and incubated on ice for 30 minutes with 1 μg ofantibody. Protein-A-sepharose was added and allowed to mix at 4° C. for60 minutes. The beads were washed 5 times in RIPA buffer before additionLaemmli buffer and boiling for 5 minutes. Immunoprecipitates were thenloaded on an SDS-10% polyacrylamide gel, blotted onto nitrocellulose andprobed with a goat anti-rabbit eIF3 antibody. Immunoreactive specieswere visualized using the Renaissance chemiluminescence system (ECL;Amersham). Affinity-purified antibodies against recombinant hPrt1 wereobtained as described in Harlow and Lane, ANTIBODIES: A LABORATORYMANUAL, Cold Spring Harbor (1988). E. coli extracts expressing hPrt1N90146 were fractionated by SDS-PAGE and transferred ontonitrocellulose. The bands containing N90146 were excised, blocked inBlotto (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.075% Tween-20, 0.5% milkpowder) and incubated with crude A-eIF3, and washed. Antibodies bound tothe membrane were eluted with 2 M glycine, I mM EGTA, pH 2.5, andneutralized by the addition of 1 M Tris-HCl, pH 8.8. To eliminatecontamination with p110 (hNip1), 50 μg of GST-pl 10 immobilizes onnitrocellulose were present during the incubation with crude α-eIF3antibodies. Western blotting was performed with antibodies at thefollowing dilutions: α-eIF3, 1:3000. α-pl70, 1:10. For western blotsperformed with the monoclonal α-pl70 antibody, horseradish-peroxidaseα-mouseIgM (Pierce) secondary antibodies were used; For α-eIF3, α-goatIgG-horseradish peroxidase; For α-hPrt1, α-goat IgG-alkalinephosphatase.

[0097] Northern Blot of hPrt1 mRNA

[0098] Total RNA from HeLa cells was isolated using Trizol (LifeTechnologies, Grand Island, N.Y.) and fractionated by electrophoresis ina 1% agarose/formaldehyde gel overnight at 40V. RNA was blotted toHybond-N+filters overnight and UV cross-linked to the membrane using UVlight. The membrane was prehybridized and hybridized under conditionsidentical to the cDNA library screening, and exposed for 24 hours to aKodak BioMax film with intensifying screen.

[0099] Far Western Blots

[0100] Partially purified FLAG-HMK hPrt1 fusion proteins (1-3 μg) were³²P-labeled using heart muscle kinase as described (Blanar, M. A., &Rutter, W. J., supra). Proteins were resolved by SDS-polyacrylamide gelelectrophoresis and blotted on PVDF membranes (Millipore) ornitrocellulose. The membranes were blocked overnight with 5% milk in HBBbuffer (25 mM HEPES-KOH, pH 7.5, 25 mM NaCl, 5 mM MgCl, 1 mM DTT), andincubated 4 hours in hybridization buffer (20 mM HEPES-KOH pH 7.5, 75 mMKCl, 2.5 mM MgCl , 0.1 mM EDTA, 1 mM DTT, 0.1% NP-40, 1% milk)containing the ³²P-labeled FLAG-HMK- or GST-FLAG-HMK-hPrt1 at 250,000cpm/ml and unlabeled purified GST at 1 μg/ml. The membranes were washed3 times with hybridization buffer and processed for autoradiography.

[0101] Results

[0102] Cloning and features of hPRT1

[0103] Expressed Sequence Tag (EST) #112738 from Human Genome Science(HGS) Inc. encodes a protein with homology to the yeast p90 eIF3subunit, Prt1. The cDNA sequence, 2 kbp in length, contained apolyadenylation signal and a short polyadenylate tail. An ATG codon waspresent at the 5′ end of the clone. However, this ATG was not precededby stop codons. It was therefore possible that EST #112738 contains anincomplete cDNA. A ³²P-labeled probe derived from the 5′ end of the ESTsequence was generated and used in a Northern analysis on HeLa cell RNA.A single RNA species migrating at 3.1 kb, hybridized with the probe. Weconcluded that I kbp was missing from EST #112738. To obtain the fulllength cDNA sequence, a human placenta λgt11 cDNA library was screenedwith the same probe used in the Northern analysis. Forty positive wererecovered and some of the cDNAs extended further upstream of the 5′-mostsequence of EST #112738. One of these clones, 3-6, contained a 3 kbpinsert with a predicted open reading frame of 873 amino acids, shown inFIGS. 1A-1D, (SEQ ID NO:2). An ATG codon, located at nucleotidepositions 97-99 downstream of the 5′ end, was preceded by an in-framestop-codon (nucleotide positions 22 to 24, shown in FIGS. 1A-1D (SEQ IDNO: 1). Thus, it is likely that clone 3-6 encodes the full length cDNA,and the first ATG constitutes the authentic initiation codon. An inframe CTG codon 24 nt upstream of the first AUG is present and couldpotentially serve as the initiation site (nucleotide positions 73-75shown in FIGS. 1A-1D (SEQ ID NO:1)). We have named the protein encodedthis cDNA hPrt1, for human-Prt1.

[0104] The cDNA sequence of hPrt1 is predicted to encode a proteincontaining a canonical RNA Recognition Motif (RRM) located between aminoacids 185 and 270 (FIGS. 1A-1D (SEQ ID NO:1)). The identification of thehPrt1 RRM is based on the consensus structural core sequences of RRMs(Birney, E., et al., Nucl. Acids Res. 21:5803-5816 (1993)), whichinclude the presence of RNP-1 and RNP-2 sequence, and hydrophobic aminoacids found at specific positions within the RRM. A BLAST search(Altschul, S. F., et al., J. Mol. Biol. 215:403-410 (1990)) with onlythe hPrt1 RRM revealed that the hPrt1 RRM is most highly related to thefourth RRM of the poly(A) binding protein PABP. No other common proteinmotifs are evident. hPrt1 is acidic, with a predicted PI of 4.8. Themiddle portion of the protein is unusually rich in tryptophan residues(close to 5% tryptophan content over 400 amino acids). Amino acidsequence comparison between human and Saccharomyces cerevisiae Prt1,reveal extensive sequence identity (31% identity, 50% homology) acrossthe entire protein except for the first 140 amino acids. The similaritybetween yeast and human Prt1 is more striking in the middle portion ofthe protein which encompasses the RRM. Several but not all of thetryptophan are conserved, suggesting that they are functionallyimportant. The amino terminus of human Prt1 is not homologous to yeastPrt1, but instead exhibits 25% identity to procollagen a chain precursorprotein (data not shown). The significance of this is not clear. ThehPrt1 protein also contains two protein kinase A, six protein kinase Cand 17 casein kinase II consensus phosphorylation sites.

[0105] hPrt1 is a part of eIF3

[0106] It is conceivable that hPrt1 is a subunit of eIF3. To prove this,an immunological characterization of hPrt1 and eIF3 was performed.First, we translated in vitro a synthetic RNA derived from the hPrt1cDNA. A single polypeptide, migrating at 116 kDa on an SDS-9%polyacrylamide gel, was obtained. The translation product co-migratedwith a 116 kDa protein in a HeLa extract that cross-reacted with α-eIF3.Thus, the size of hPrt1 is similar to one of the eIF3 subunits. Next, wetested the ability of a polyclonal α-eIF3 antibody to recognize hPrt1.To this end, we expressed hPrt1 fused to the hemagglutinin (HA)epitope-tag in HeLa cells using a recombinant vaccinia virus expressionsystem (Fuerst et al., supra). Extracts from infected cells were,blotted onto nitrocellulose and probed with a polyclonal α-eIF3antibody. eIF3 subunits (pl70 and p15) in extracts from cellstransfected with the parental vector (pACTAG-2; Charest et al., Biochem.J. 308: 425-432 (1995)) or pACTAG-hPrt1 were readily identifiable. A 125kDa protein that cross-reacts with α-eIF3 was present in extracts fromcells transfected with pACTAG-hPrt1 but not in cells transfected withthe vector alone. To confirm the identity of this protein as HA-hPrt1,immunoprecipitations using α-HA antibody were performed and the productsprobed with α-eIF3 antibody. The immunoprecipitated HA-hPrt1 co-migratedwith the 125 kDa polypeptide, and cross-reacted with the α-eIF3antibody. The slower mobility of HA-hPrt1 relative to hPrt1 is probablydue to the three HA epitopes present in the fusion protein.Immunoprecipitates from cells transfected with the parental vector orwith a vector encoding HA-La autoantigen failed to cross-react withα-eIF3. We conclude that hPrt1 is recognized by an antibody directedagainst eIF3. Finally we wished to determine whether antibodies directedagainst hPrt1 could recognize a 116 kDa polypeptide in purified humaneIF3. Attempts to generate antibodies against hPrt1 in rabbits failed.To circumvent this problem, affinity-purified hPrt1-specific antibodiesfrom α-eIF3 antisera were prepared from crude eIF3 antibodies, using abacterially expressed hPrt1 fragment. These antibodies recognized aprotein migrating at approximately 116 kDa in a highly purified humaneIF3 preparation and in HeLa extracts, and did not cross-react withhNip1 a 110 kDa protein also recently shown to be an eIF3 component (seethe discussion below). Together with the previous data, theseexperiments strongly suggest that hPrt1 is the 115 kDa subunit of eIF3.

[0107] hPrt1 interacts directly with the p170 subunit of eIF3

[0108] To further substantiate the finding that hPrt1 is a subunit ofhuman eIF3, we examined the possibility that hPrt1 interacts directlywith one or more eIF3 subunits. To this end, hPrt1 was tagged with aFLAG peptide linked to a heart muscle kinase site (FLAG-HMK) or fused toa glutathione-S-transferase-FLAG-HMK sequence (GST-FLAG-HMK). We optedto use fragments of hPrt1 rather than the full length protein due to thelow yield and extensive degradation of full length hPrt1 in E. coli. Theproteins were purified using a FLAG antibody or glutathione-sepharoseresin, and were ³²P-labeled with heart muscle kinase. The labeledproteins were then used to detect interacting proteins by the FarWestern assay with HeLa cytoplasmic extracts, rabbit reticulocyte lysateand different preparations of eIF3. Two of the probes, GST N255 (aminoacids 1-255 of hPrt1 shown in FIGS. 1A-1D (SEQ ID NO:2)) and N90146(amino acids 147-873 shown in FIGS. 1A-1D (SEQ ID NO:2)), interactedwith a 170 kDa protein in HeLa and rabbit reticulocyte lysate. The hPrt1probes reacted with a 140 kDa protein in eIF3 preparation 1 and 140 and170 kDa polypeptides in eIF3 preparation 2. A ³²P-labeled probeconsisting only of a GST-HMK fusion did not recognize any proteins (datanot shown). The 170 kDa protein in HeLa, rabbit reticulocytes and eIF3preparation 2 could the largest subunit of eIF3, pl70. This protein issensitive to degradation, and the two eIF3 preparations used here differby the extent of pl70 proteolysis. An immunoblot using a monoclonalantibody directed against p170 (Mengod, G., & Trachsel, H., Biochem.Acta 825:169-174 (1985)) revealed the extent of p170 degradation in theeIF3 preparations, and clearly shows that a 140 kDa degradation productof p170 is present in preparation 1, and to a lesser extent inpreparation 2. This experiment demonstrates that hPrt1 interactsdirectly with the p170 subunit of eIF3. No other eIF3 subunits wererecognized by the hPrt1 probes in this assay (data not shown).

[0109] The fact that both the N90146 and N255 fragments of hPrt1 reactedwith p170 suggest that the site of protein-protein interaction islocated between amino acids 147 and 255 (FIGS. 1A-1D (SEQ ID NO:2)).This segment of hPrt1, which encompasses most of the RRM, was assessedfor its ability to interact with p170 independently of other sequences.A fragment containing amino acids 147 to 255 of hPrt1, used as a probein a Far Western assay, indeed interacted with p170. To furtherdelineate the interaction site, a fragment consisting of amino acids147-209 (FIGS. 1A-1D (SEQ ID NO:2)) was tested, and failed to interactwith p170 (data not shown). This suggests that a portion of the RRM iscrucial for the association between hPrt1 and p170.

[0110] Discussion

[0111] The present invention provides a human cDNA that with homology tothe yeast eIF3 subunit, Prt1. In vitro translation of hPrt1 RNA yieldeda polypeptide of 116 kDa that co-migrated with one of the eIF3 subunits.Immunological characterization revealed that hPrt1 cross-reacts withÂ-eIF3 and that affinity-purified Â-hPrt1 antibodies recognize apolypeptide of approximately 120 kDa in highly purified eIF3. Thus, adirect interaction between hPrt1 and the p170 subunit of mammalian eIF3has been demonstrated. Based on these data, the inventors conclude thathPrt1 corresponds to the second largest subunit of eIF3, p115. Theimmunoprecipitates of HA-hPrt1 did not contain other eIF3 subunits. Itis likely that HA-hPrt1 does not incorporate well into the endogenouseIF3 because of the stability of the complex. Alternatively, the HA-tagmay hinder association of HA-hPrt1 with eIF3.

[0112] Recently, Hershey and co-workers have isolated a human cDNApredicted to encode a 110 kDa protein which showed homology to the yeastNip1 protein. Although Nip1 is not present in yeast eIF3 complexes, thehuman homologue is part of mammalian eIF3. The data disclosed hereinsuggests that mammalian eIF3 contains two subunits that migrate atapproximately 115 kDa. Examination of various rat or rabbit eIF3preparations resolved on SDS-polyacrylamide gel electrophoresis show twopolypeptides migrating at this position (Behlke, J. et al., Eur. JBiochem. 157:523-530 (1986); Meyer, L. et al., Biochemistry 21:4206-4212(1982)). Thus, the mammalian eIF3 complex consists of at least 9polypeptides: p170, p116 (hPrt1), p110 (hNip1), p66, p47, p44, p40, p36and p35.

[0113] Mutations in the PRT1 gene of Saccharomyces cerevisiae impairtranslation initiation in vivo at 37° C. (Hartwell, L. and McLaughlin,C., J. Bacteriol. 96:1664-1671 (1968); Hartwell, L. and McLaughlin, C.,Proc. Natl. Acad. Sci. USA 62:468-474 (1969)). One of the mutants,prt1-1, does not promote binding of the ternary complexeIF2-GTP-tRNAimet to the 40S ribosomal subunit (Feinberg, B., et al., J.Biol. Chem. 257:10846-10851 (1982)). Evans et al. (1995) identified sixmutations in PRT1 which impair translation initiation (Evans, D. R. H.,et al., Mol. Cell. Biol. 15:4525-4535 (1995)). Two of these mutationsalter amino acids that are conserved between yeast and human Prt1. HumanPrt1, when expressed in the prt1-1 yeast strain, was unable to rescuethe temperature sensitive phenotype (N. Methot, unpublished data). Thiswas somewhat surprising since yeast eIF3 functions in a mammalianmethionyl-puromycin assay system (Naranda, T., et al., J. Biol. Chem.269:32286-32292 (1994)). Methionyl-puromycin synthesis is dependent onthe binding of the ternary complex to the 40S ribosome, requires onlywashed ribosomes, tRNAimet, eIF1A, eIF2, eIF3, eIF5 and eIF5A (Benne,R., et al., Meth. Enzymol. 60:15-35), but does not measure MRNA bindingto the ribosome. It is clear that yeast eIF3 can replace mammalian eIF3for some, but not all normal eIF3 functions, and that hPrt1 is unable tofulfill all the roles of yeast Prt1. One of the reasons why hPrt1 wasunable to replace Prt1 in vivo is that it may not incorporate into theyeast eIF3 complex. A Far Western analysis on yeast extracts using thehPrt1 N255 fragment did not reveal any interacting proteins.

[0114] Both yeast and human Prt1 contain near their amino terminus anRNA recognition motif (RRM; residues 185-270 in hPrt1, shown in FIGS.1A-1D (SEQ ID NO:2)). The RRM contains the sequence elements that areresponsible for specific protein-protein interactions with the p170subunit of eIF3. It is unlikely that the interaction between hPrt1 andp170 is mediated through RNA since hPrt1 was unable to bind aradiolabeled RNA probe as measured by UV photocrosslinking andNorthwestern assays (N. Methot, unpublished observations). Further,treatment of the FLAG-HMK N90146 probe and the nitrocellulose membranewith RNase A did not reduce the intensity of the interaction with p170(N. Methot, unpublished data). It is possible that the RRM is functionalas an RNA binding module only within the eIF3 complex, and that its RNAbinding activity and specificity are modulated by pl70. Precedents forprotein-protein interactions altering the RNA binding activity of anRRM-containing protein exist. The spliceosomal protein U2B″ is unable onits own to distinguish between the U1 and U2 snRNAs, but will bindspecifically to U2″ snRNA in the presence of the U2A′ protein (Scherly,D., Nature (London) 345:502-506 (1990); Scherly, D., et al., EMBO J.9:3675-3681 (1990)). U2A′ and U2B″ associate in the absence of RNA, aninteraction which is mediated by the RRM (Scherly, D., et al., EMBO J.,supra). The major RNA binding protein of yeast eIF3 is the p62 subunit(Naranda, supra). It has been previously shown that the p170 subunit ofeIF3 interacts directly with eIF4B (Methot, N., et al., Mol. Cell. Biol.in press (1996)). eIF4B and hPrt1 do not appear to interact with thesame sites on p 170 since hPrt1 reacts very strongly with the 140 kDadegradation product of p 170, while eIF4B does not. The numerousprotein-protein interactions involving p170 suggest that this proteinmay serve as a scaffold for both the assembly of the eIF3 complex andfor the binding of the mRNA to the ribosome.

Example 2

[0115] Cloning of cDNAs

[0116] The CDNA #20881 was obtained from a human embryo brain cDNAlibrary by random cloning. A human placenta cDNA library in λgt11 wasscreened with a fragment (nucleotide positions, 473 to 1200, shown inFIGS. 2A-2E (SEQ ID NO:3)) of CDNA #20881. 5′-RACE (rapid amplificationof CDNA ends, Invitrogen Life Technologies) was performed with HeLa poly(A)+RNA and sequence specific primers (594 to 614 and 643 to 664 shownin FIGS. 2A-2E (SEQ ID NO:3)) according to the manufacturer'sinstructions.

[0117] Construction of Plasmids

[0118] To generate the carboxyl (C)-terminally HA-tagged cDNAs, anantisense primer composed of the sequences encoding the C-terminal sixamino acids of p97 followed by the HA epitope peptide, YPYDVPDYAG (SEQID NO:13), and nucleotides corresponding to the Xho I site was used forpolymerase chain reaction (PCR) with a sense primer (nucleotides 2527 to2549 shown in FIGS. 2A-2E (SEQ ID NO:3)). pcDNA3, which has a humancytomegalovirus (CMV) and T7 RNA polymerase promoters, was used as anexpression vector for most of the experiments. pcDNA3-4-1-A(HA) andpcDNA3-6-4-A(HA) contain the corresponding p97 cDNA sequences downstreamof nucleotide positions 12 and 30 in FIGS. 2A-2E (SEQ ID NO:3),respectively. pcDNA3-ATG-A(HA) contains the sequence downstream ofnucleotide 473 and a part of the sequence of transcription factor BTEB(-15 to 10, including the initiator ATG) (Imataka, H., et al., EMBO J.11:3633-3671 (1992)).

[0119] For an N-terminally HA-tagged construct, the initiator ATG codonand three copies of the HA sequence (Mader, S., et al., Mol. Cell. Biol.15:4990-4997 (1995)) were inserted into pcDNA3 to generate the parentalvector, pcDNA3-HA. A PCR amplified fragment from the GTG initiationcodon to a SacI site (nucleotide 600) was ligated to a fragment fromSacI to the 3′-terminus of cDNA #20881 to construct pcDNA3-HA-p97. AnEcoRI fragment of the human eIF4G cDNA (kindly provided by Dr. Rhoads;Yan, R., et al., J. Biol. Chem. 267:23226-23231 (1992)) was used toconstruct pcDNA3-HA-eIF4G. HA-La was inserted into pcDNA3 to obtainpcDNA3-HA-La.

[0120] For expression of non-tagged p97, a fragment from a BamHIrestriction site (nucleotide 172 (FIGS. 2A-2E (SEQ ID NO:3)) to the3′-terminus in which the initiator GTG had been mutated into ATG, wasinserted into pcDNA3 to generate pcDNA3Bam-ATGp97. A point mutation (GTGto ATG or to GGG) was introduced using a conunercial kit (Amersham). Toconstruct pcDNA3-eIF4G, the EcoRi fragment of eIF4G cDNA was insertedinto pcDNA3.

[0121] The poliovirus internal ribosome entry site (IRES) was insertedinto pSP72, which contains the T7 RNA polymerase promoter, to generatepSP72IES. For expression of p97 or eIF4G in a cap-independent manner,the Bam-ATGp97 and the EcoRI fragment of eIF4G cDNA were inserteddownstream of the IRES to construct pSP72IRES-p97 and pSP72IRES-eIF4G.

[0122] Transient Transfection

[0123] HeLa cells were infected with recombinant vaccinia virus vTF7-3(Fuerst et al., supra), and then transfected with plasmids (5 μg) usingLipofectin (Invitrogen Life Technologies, Carlsbad, Calif.). Forexpression in COS-1 cells, plasmids (10 μg) were transfected byelectroporation (Bio-Rad Gene PulserII, 1200V, 25mF).

[0124] Immunoprecipitation

[0125] After transfection, HeLa and COS-1 cells were cultured for 16hours and 48 hours, respectively, and then labeled with [³⁵S]methionine(100 μCi/ml) for 1 hour in 3 cm dishes. Cells were lysed in0.5 ml buffer A (150 mM NaCl, 1% NP-40, 0.1 % deoxycholate, 1 mMphenylmethylsulfonyl fluoride (PMSF), 1mM dithiothreitol (DTT), 50 mMTris-HCl, pH 7.4). After centrifugation, the supernatant was mixed with2 μg of anti- HA antibody (12CA5) for 6 hours in the cold room at 4° C.Protein G sepharose (30 ml of 50% slurry) was added and the mixture wasincubated for 2 hours. After washing with buffer A (1 ml, three times),immunoprecipitates were collected by centrifugation and proteins wereeluted with Laemmli buffer for SDS-10% polyacrylamide gelelectrophoresis (SDS-PAGE). For co-immunoprecipitation experiments,transfected HeLa cells (6 cm dish) were lysed in 1 ml buffer B (100 mMKCl, 0.5 mM EDTA, 20 mM HEPES-KOH pH 7.6, 0.4% NP-40, 20 % glycerol, 1mM DTT, 1 mM PMSF, 5 μg/ml pepstatin, 5 μg/ml leupeptin). Aftercentrifugation, an aliquot (0.5 ml) was mixed with anti-HA antibody (2μg). Immuprecipitates were washed with buffer B (1 ml, three times), andresolved by SDS-10% PAGE, except for eIF4E experiments where 12.5%polyacrylamide gels were used.

[0126] Western Blotting and Antibodies

[0127] Immunoprecipitates or cell extracts (60 μg protein) were resolvedby SDS-PAGE and transferred to Immobilon polyvinylidene difluoridemembrane (Millipore). Protein bands were visualized by chemiluminescence(Amersham). Quantification was done with a laser densitometer (LKB).

[0128] Anti-p97 antibodies: The peptide, SDETDSSSAPSKEQ (called INT,amino acids 788 to 802, shown in FIGS. 2A-2E (SEQ ID NO:4)) conjugatedwith keyhole limpet hemacyanin was used to raise anti-peptide(INT)antibody in rabbits. For absorption experiments, serum was pre-incubatedwith the INT peptide (10 μg) on ice for 1 hour, and was used for Westernblotting. A fusion protein GST-C-terminus, glutathione-S-transferaselinked to the peptide ETAEEEESEEEAD (amino acids, 895 to 907, shown inFIGS. 2A-2E (SEQ ID NO:4)) was produced in E. coli for immunization inrabbits. The resulting serum was passed through a GST or GST-C-terminuscolumn for adsorption.

[0129] CAT Assay and RNase Protection Assay

[0130] Chloramphenicol acetyltransferase (CAT) assay was performed asdescribed (Gorman, C. M., et al., Mol. Cell. Biol. 2:1044-1051 (1982)).RNase protection assay was done as described (Imataka, H., et al., EMBOJ. 11:3633-3671 (1992)) with modifications as follows: as an internalcontrol, in vitro synthesized unlabeled BTEB RNA and radiolabeledantisense RNA to BTEB sequence (Imataka, H., et al., EMBO J.11:3633-3671 (1992)) were mixed with antisense CAT probe. Intensities ofthe CAT and BTEB signals were quantified by Phosphorimager BAS 2000. Theamount of CAT mRNA was normalized to that of the internal control BTEBRNA. Translation activity was calculated by dividing CAT activity by thenormalized CAT mRNA amount.

[0131] In Vitro Transcription and Translation

[0132] Capped RNA was synthesized by T7 RNA polymerase in the presenceof the cap analogue, m⁷GpppG. Rabbit reticulocyte lysate (25 μl, finalvolume) was programmed with 0.2 μg of mRNA in the presence of[³⁵S]methionine (20 μCi) according to the manufacturer's recommendation.

[0133] Binding Assay for in Vitro Translated Factors

[0134] Following translation, five microliters of the lysate wasincubated on ice for 30 minutes with anti-FLAG resin (20 pi) to whichFLAG-eIF4E or FLAG-eIF4A had been bound. After washing with buffer Cconsisting of 50 mM Tris-HCl (pH, 7.5), 1 mM EDTA, 0.15 M NaCl and 0.1 %NP-40 (1 ml, three times), bound proteins were eluted with 40 μl ofbuffer C containing 100 μg/ml FLAG peptide. pAR(DRI)[59/60] (Blanar, M.A., & Rutter, W. J., supra) was used to express FLAG-eIF4E (Pause, A.,et al., Nature 371:762-767 (1994)) and FLAG-eIF4A in E. coli.

[0135] Induicible Expression of p97

[0136] p97 cDNA (nucleotides 36 to 3810 in FIGS. 2A-2E (SEQ ID NO:3)),in which the initiator GTG codon was converted to ATG, was inserted intoa tetracycline-dependent expression vector, pRep9-CMVt (Beauparlant, P.,et al., J. Biol. Chem. 271:10690-10696 (1996)) to constructpRep9-CMVt-p97. An NIH3T3-derived cell line, S2-6 (Shockett, P., et al.,Proc. Natl. Acad. Sci. USA 92:6522-6526 (1995)) was transformed withpRep9-CMVt (control) or with pRep9-CMVt-p97 using G418 (400 μg/ml). S2-6and the established transformants were maintained in the presence of 1μg tetracycline/ml. To induce p97 expression, cells were cultured inmedium without tetracycline for 40 hours. After induction, cells wereprocessed for Western blotting or labeled with [^(2, 3, 5-3)H] leucine(20 μCi/ml). Cells were lysed with buffer B and extracts (20 μg protein)were applied to filter paper. After washing with trichloroacetic acid(5%), radioactivity remaining on the paper was counted.

[0137] Results

[0138] The GUG-initiated open reading frame encodes a variant of eIF4G

[0139] A human CDNA clone #20881 (hereafter called clone A, nucleotidepositions 473 to 3820 in FIGS. 2A-2E (SEQ ID NO:3)) was found to possessan open reading frame (ORF) encoding a protein (850 amino acids) similarto eIF4G. RNA synthesized from clone A produced no protein in areticulocyte lysate translation system (data not shown). The ORF ofclone A had no translation initiator ATG; the first in-frame ATG(nucleotide positions 925-927, shown in FIGS. 2A-2E (SEQ ID NO:3)) isunlikely to be the initiation codon, since there are eleven upstreamATGs which are out of frame between nucleotide positions 473 and 927.Thus, upstream sequences which could provide the translation initiatorare lacking from clone A. The 3′-terminus is complete because of thepresence of the poly (A) signal and a poly (A) tail (FIGS. 2A-2E (SEQ IDNO:3)). To obtain a full length cDNA, we screened a human placenta cDNAlibrary with a 5′ sequence of clone A. 14-1, the longest clone obtained,starts at nucleotide position 12 in FIGS. 2A-2E (SEQ ID NO:3) and thesequence was extended by 11 nucleotides by 5′-RACE. The longest sequence(3820 nucleotides) (FIGS. 2A-2E (SEQ ID NO:3)) is close to full-length,since Northern blotting showed that the mRNA is approximately 3.8 kb inlength. The mRNA is expressed in every tissue and cell line examined,implying a fundamental role of the protein in cells. The first ATG ofthe extended ORF is the same as that identified in clone A (nucleotidepositions 925-927 shown in FIGS. 2A-2E (SEQ ID NO:3)) and there arein-frame stop codons at nucleotide positions 178 and 205. The sequence4-1-A (nucleotides 12 to 3820 shown in FIGS. 2A-2E (SEQ ID NO:3)) wasused for further studies.

[0140] To determine the capacity of the full length cDNA to encode aprotein, a modified cDNA, 4-1-A(HA), in which the hemagglutinin (HA)epitope was fused to the C-terminus of the ORF, was transfected intoCOS-1 and HeLa cells. Western blotting and inununprecipitation withanti-HA antibody demonstrated that a 97 kDa protein (called, p97-HA) wassynthesized. Transfection of a truncated cDNA, ATG-A(HA), in which anartificial ATG was inserted in frame to initiate the ORF of clone A,yielded a shorter polypeptide than p97-HA. The apparent molecular massof this shorter protein is 95 kDa, which is close to the expected sizefrom the sequence of clone A (97 kDa, ORF of clone A plus HA). Theseresults indicate that the full-length cDNA encodes a protein which islarger than that encoded by the ORF starting from position 473. Oneexplanation for this is that translation of this protein starts at anon-AUG initiator 5′ upstream of clone A. Although there is one ATGtriplet in the 5′ upstream region (nucleotide positions 21-23 shown inFIGS. 2A-2E (SEQ ID NO:3)), it is predicted to encode a smallpolypeptide (16 amino acids), and is out of frame of clone A ORF.Furthermore, transfection of 6-4-A(HA), which does not contain this ATG(FIGS. 2A-2E (SEQ ID NO:3)), produced a protein indistinguishable fromp97-HA.

[0141] We predicted that GTG at nucleotide positions 307 to 309 (FIGS.2A-2E (SEQ ID NO:3)) is the translation initiator, since it couldpotentially start an ORF that encodes a polypeptide of about 100 kDa,and the nucleotide sequence flanking this triplet (GCCGCCAAAGUGGAG,nucleotides 298-312 in FIGS. 2A-2E (SEQ ID NO:3)) is similar to theconsensus sequence for non-AUG initiators (Boeck, R. & Kolakofsky, D.EMBO J. 13:3608-3617 (1994); Grunert, S. & Jackson, R., EMBO J.13:3618-3630 (1994)). To test this possibility, we mutated the GTG intoGGG or ATG in the 4-1-A(HA) construct, and transfected the DNA into HeLacells. The ATG mutant yielded 4 fold more p97-HA protein than the wildtype from similar amounts of RNA, while the GGG mutant failed to producethe protein. In vitro translation experiments confirmed the in vivoresults. When the 4-1-A(HA) RNA was translated in a rabbit reticulocytelysate, p97-HA was synthesized as a single product. A point mutation(GUG to GGG) abolished translation, while a mutation to AUG increasedtranslation of p97-HA by 2 fold. From these data, we conclude thattranslation of p97 mRNA exclusively starts at the GUG codon (positions307-309 in FIG. 2A-2E (SEQ ID NO:3)) to encode a polypeptide of 907amino acids (FIGS. 2A-2E (SEQ ID NO:3)). This mode of translation isapparently not specific to the human p97 mRNA, since the cDNA sequenceof the mouse p97 homologue also lacks an initiator ATG, and the GTGcodon is conserved.

[0142] To verify the presence of p97 protein in cells, we used twodifferent antisera raised against the same p97 peptide sequences as wereused above in Western blotting. Experiments were performed with extractsfrom a mouse cell line Neuro2A, since anti-GST-C serum pre-incubatedwith GST or GST-C detects polypeptides between 95 and 100 kDa withextracts from primate cell lines, including HeLa and COS-1, and thesenon-specific bands obscure the p97 band (data not shown).Anti-GST-C-terminal peptide antiserum detected two bands with apparentmolecular masses of 97 and 65 kDa, both of which disappeared when theserum was absorbed with the antigen. The 65 kDa polypeptide is likely across-reacting material, since another serum, anti-peptide (INT) serum,did not detect this band. In contrast, the 97 kDa band was also detectedby the latter serum, and disappeared by treatment of the serum with thepeptide (INT). Thus, the 97 kDa polypeptide is the only commonpolypeptide that is specifically recognized by the two differentantisera. To further substantiate the authenticity of the 97 kDapolypeptide, we expressed non-tagged p97 from a cDNA. HeLa cells wereemployed for transfection because of their better transfectionefficiency. The amount of p97 was increased by 4 fold followingtransfection with a non-tagged p97 expression plasmid,pcDNA3-Bam-ATGp97. Therefore, clearly, p97 is translated from theendogenous MRNA.

[0143] p97 binds to eIF4A and eIF3, but not to eIF4E

[0144] Alignment of human p97 and eIF4G amino acid sequences revealsthat p97 exhibits 28% identity and 36% similarity to the C-terminal twothirds of eIF4G. The N-terminal third of eIF4G, to which eIF4E binds(Lamphear, B., supra; Mader, S., et al., supra), bears no similarity top97. Therefore, no canonical eIF4E-binding site (Mader, S., et al.,supra) is found in p97. Lamphear, B., et al., supra showed that theC-terminal two thirds of the poliovirus protease-cleaved eIF4G containsthe binding sites for eIF4A and eIF3. Thus, it is predicted that p97would bind to eIF4A and eIF3, but not to eIF4E. To examine this,HA-tagged p97 and eIF4G were expressed in HeLa cells, and cell extractswere immunoprecipitated with anti-HA antibody. The immunoprecipitateswere assayed by Western blotting for eIF4A, eIF3 and HA-tagged proteinexpression. Both eIF4A and eIF3 were co-precipitated with p97 and eIF4G,while an RNA binding protein, La autoantigen (Chambers, J. C., et al.,J. Biol. Chem. 263:18043-18051 (1988)) failed to precipitate eitherfactor.

[0145] The light chain of the HA-antibody co-migrates with eIF4E onSDS-PAGE, rendering detection of immunoprecipitated eIF4E difficult. Tocircumvent this problem, we co-expressed FLAG-tagged eIF4E, whichmigrates slower than non-tagged eIF4E, with HA-tagged proteins. Cellextracts were immunoprecipitated with anti-HA, and theimmunoprecipitates were examined by Western blotting for eIF4E andHA-tagged proteins. No detectable FLAG-eIF4E was co-precipitated withp97 or with La, while eIF4G was able to precipitate FLAG-eIF4E.

[0146] To further substantiate these results, p97 or eIF4G wassynthesized in vitro and mixed with bacterially expressed FLAG-eIF4E orFLAG-eIF4A bound to the anti-FLAG resin. Proteins bound to the anti-FLAGresin were eluted with the FLAG peptide. p97 and eIF4G specificallybound to eIF4A. In contrast, binding of p97 to eIF4E was not detectable,while interaction between eIF4G and eIF4E was evident. La failed to bindto either resin. We were not able to perform similar experiments foreIF3, since it consists of multi-subunits, and it is not known whichsubunit(s) interact(s) with eIF4G or p97. Thus, we conclude that p97forms a protein complex which includes eIF4A and eIF3, but excludeseIF4E.

[0147] p97 suppresses cap-dependent and cap-independent translation

[0148] Ohlmann, T., et al., EMBO J. 15:1371-1382 (1996) showed that theC-terminal two thirds of eIF4G supports cap-independent translation.Based on its homology to eIF4G, p97 might also promote cap-independenttranslation. To explore this possibility, we expressed p97 and eIF4G inHeLa cells together with a reporter CAT (chloramphenicolacetyltransferase) mRNA, whose ORF is preceded by theencephalomyocarditis virus internal ribosome entry site (EMCV-IRES).Translation of EMCV-IRES-CAT mRNA was repressed 2 fold by expression ofp97. In contrast, eIF4G stimulated cap-independent translation by 2 fold(these experiments were repeated 4 times with less than 10% variationbetween the results). Moreover, eIF4G relieved the p97-inducedrepression of translation, indicating that p97 inhibits cap-independenttranslation by competing with eIF4G. To study the effect of p97 oncap-dependent translation, CAT mRNA was used as the reporter. Similarlyto its effect on cap-independent translation, p97 inhibitedcap-dependent translation by 2 fold and the inhibition was partiallyrelieved by co-expression of eIF4G.

[0149] To study how p97 expression affects overall protein synthesis incells, we established a cell line that expresses p97 under atetracycline-regulatable promoter (Beauparlant, P., et al., J. Biol.Chem. 271:10690-10696 (1996)). Withdrawal of tetracycline from themedium increased the amount of p97 about 4-fold without noticeablechange of the amounts of eIF4G, eIF4E or eIF4A. Overexpression of p97decreased the rate of protein synthesis by 20 to 25% as determined byincorporation of [³H] leucine. We performed similar labeling experimentswith [³⁵S] methionine and obtained essentially similar results (data notshown). These functional assays, combined with the binding results,suggest that p97 is a general suppressor of translation by forming atranslationally inactive protein complex that includes eIF4A and eIF3,but excludes eIF4E.

[0150] Discussion

[0151] The present invention further provides a new translationalregulator, p97, which is homologous to the C-terminal two thirds ofeIF4G. This region of eIF4G contains binding sites for eIF4A and eIF3,while the binding site for eIF4E is present in the N-terminal third ofthe protein (Lamphear, B., et al., supra; Mader, S., et al., supra). p97binds to eIF4A and eIF3, but not to eIF4E. While the C-terminal twothirds fragment of eIF4G is able to support translation initiation fromthe internal ribosome entry site (IRES) of hepatitis C virus andTheiler's murine encephalomyelitis virus (Ohlmann, T., et al., supra),p97 inhibits EMCV-IRES dependent translation. It is unlikely that theopposite effects on translation are due to different IRES elements,since poliovirus IRES-mediated translation is promoted by the C-terminusof eIF4G, while transient expression of p97 repressed translation ofpoliovirus IRES-CAT MRNA. Thus, it is likely that p97 generally inhibitsIRES-dependent translation, while the C-terminal two thirds of eIF4Ggenerally supports IRES-independent translation.

[0152] p97 most likely inhibits translation by sequestration of eIF4Aand eIF3, thus keeping these proteins from interacting with eIF4G. eIF4Ais absolutely required for cap-dependent and cap-independent translation(Pause, A., et al., supra) and eIF3 is essential for recruitment ofribosomes to mRNA (Pain, V. M., Eur. J. Biochem. 236:747-771 (1996)).p97 and eIF4G are likely to compete for eIF4A and eIF3 binding since,expression of eIF4G relieves p97-dependent repression of translation.This model of translational inhibition is reminiscent of the mechanismby which eIF4E-binding proteins inhibit translation. 4E-BP-1 competeswith eIF4G for binding to eIF4E, and thereby inhibits formation of thecomplete eIF4F complex (Haghighat, A., et al., EMBO J. 14:5701-5709(1995)). While 4E-BP-1 and eIF4E were reported to exist in reticulocytelysate at an approximately 1:1 molar ratio (Rau, M., et al, J. Biol.Chem. 271:8983-8990 (1996)), the present inventors could not determinethe molar ratio of p97 to other translation factors because of thedifficulty in obtaining pure recombinant protein. The relative ratios ofeIF4A, eIF4G (Duncan, R., et al., J. Biol. Chem. 262:380-388 (1987)) andeIF3 (Meyer, L. J., et al., supra; Mengod, G. & Trachsel, H. Biochem.Biophys. Acta 825:169-174 (1985)) to ribosomes in HeLa cells have beenreported to be 3, 0.2 and 0.5, respectively.

[0153] Plants have two different eIF4F complexes. One is a complex oftwo polypeptides, p220 and p26, which are homologues of mammalian eIF4Gand eIF4E. The other complex, called eIF(iso)4F, consists of p28,another homologue of mammalian eIF4E, and p82 (Browning, K. S., et al.,J. Biol. Chem. 265:17967-17973 (1990); Allen, M., et al., J. Biol. Chem.267:23232-23236 (1992)). p82 exhibits significant sequence similarity tohuman eIF4G (Allen, M., et al., supra), and the binding site for eIF4Eis present in the N-terminus of p82 (Mader, S., et al., supra).eIF(iso)4F, like eIF4F, stimulates translation in vitro (Abramson etal., J. Biol. Chem. 263:5462-5467 (1988)). Yeast also has two genesencoding eIF4G homologues, TIF4631 and TIF4632 (Goyer, C., et al., Mol.Cell. Biol. 13:4860-4874 (1993)). Although both contain an eIF4E-bindingsite (Mader, S., et al., supra), there seems to be a functionaldifference between two proteins, since TIF4631-disrupted strainsexhibited a slow-growth, cold sensitive phenotype, while disruption ofTIF4632 failed to show any phenotype. Double gene disruption engenderedlethality (Goyer, C., et al., Mol. Cell. Biol. 13:4860-4874 (1993)). Itis possible that p97 has evolved from eIF4G to become a repressor bylosing the binding site for eIF4E.

[0154] Why is GUG employed instead of AUG?

[0155] p97 mRNA has no initiator AUG and translation exclusively startsat a GUG codon. The nucleotide sequence surrounding the initiator GUG isGCCAAAGUGGAG (nucleotides 301-312 in FIGS. 2A-2E (SEQ ID NO:3)), whichmatches the consensus rule that purines are favorable at positions -3and +4 (the first nucleotide of the initiation codon is defined as +1,shown herein as nucleotide 307 in FIGS. 2A-2E (SEQ ID NO:3)) (Kozak, M.,J. Cell. Biol. 108:229-241 (1989)). More importantly, p97 mRNA hasadenine at the +5 position. Translation starting at a non-AUG isefficient when the second codon is GAU, where G at +4 and A at +5 aremore important than U at +6 (Boeck, R. & Kolakofsky, D. EMBO J.13:3608-3617 (1994); Grunert, S. & Jackson, R., EMBO J. 13:3618-3630(1994)).

[0156] Several important regulatory genes including c-myc (Hann, S. R.,et al., Cell 52:185-195 (1988)), int-2 (Acland, P., et al., Nature343:662-665 (1990)), pim-1, FGF-2 (Florkiewicz, R. Z. & Sommer, A.,Proc. Nati. Acad. Sci. USA 86:3978-3981 (1989)) and WT-1 (Bruening, W. &Pelletier, J., J. Biol. Chem. 271:8446-8454 (1996)) have non-AUGinitiators in addition to a downstream and in-frame AUG initiationcodon, so that non-AUG initiated translation generates amino-terminallyextended proteins. Some of the extended proteins show differentintracellular localization than their shorter counterparts (Acland, P.,et al., Nature 343:662-665 (1990); Bugler, B., et al., Mol. Cell. Biol.11:573-577 (1991)). In contrast, multiple products are not produced fromp97 MRNA, since the GUG is the only initiator. Translation initiation atCUG of c-Myc mRNA was enhanced, when culture medium was deprived ofmethionine (Hann, S. R., et al., Genes & Dev. 6:1229-1240 (1992)). ForFGF-2, eIF4F seems to activate utilization of CUG more than that of AUG(Kevil, C., et al., Oncogene 11:2339-2348 (1996)). The expression of p97might also be translationally controlled.

[0157] What is the biological significance ofp97?

[0158] p97 is a putative modulator of interferon- -induced programmedcell death. Also, apoptosis has been shown to be affected by proteinsynthesis inhibitors (Martin, D. P., et al., J. Cell Biol. 106:829-844(1988); Ledda-Columbano, G. M., et al., Am. J. Pathol. 140:545-549(1992); Polunovsky, V. A., et al., Exp. Cell Res. 214:584-594 (1994)),and overexpression of eIF4E in NIH3T3 cells prevents apoptosis inducedby serum depletion. Further, p97 mRNA is heavily edited, whenapolipoprotein B mRNA-editing protein is overexpressed in the liver oftransgenic mice, suggesting that the amount of p97 might be controlledby an editing mechanism.

Example 3

[0159] Cloning and Expression of hPrt1 and p9 7 Proteins in aBaculovirus Expression System

[0160] In this illustrative example, the plasmid shuttle vector pA2 GPis used to insert the cloned DNA encoding the hPrt1 and p97 proteins,both of which lack naturally associated secretory signal (leader)sequences, into a baculovirus to express the mature proteins, using abaculovirus leader and standard methods as described in Summers et al.,A Manual of Methods for Baculovirus Vectors and Insect Cell CultureProcedures, Texas Agricultural Experimental Station Bulletin No. 1555(1987). This expression vector contains the strong polyhedrin promoterof the Autographa californica nuclear polyhedrosis virus (AcMNPV)followed by the secretory signal peptide (leader) of the baculovirusgp67 protein and convenient restriction sites such as BamHI, Xba I andAsp718 . The polyadenylation site of the simian virus 40 (“SV40”) isused for efficient polyadenylation. For easy selection of recombinantvirus, the plasmid contains the beta-galactosidase gene from E. coliunder control of a weak Drosophila promoter in the same orientation,followed by the polyadenylation signal of the polyhedrin gene. Theinserted genes are flanked on both sides by viral sequences forcell-mediated homologous recombination with wild-type viral DNA togenerate viable virus that expresses the cloned polynucleotide.

[0161] Many other baculovirus vectors could be used in place of thevector above, such as pAc373, pVL941 and pAcIM1, as one skilled in theart would readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, in Luckow et al.,Virology 170:31-39.

[0162] The cDNA sequence encoding the hPrt1 or p97 proteins in thedeposited clones, containing the AUG initiation codon is amplified usingPCR oligonucleotide primers corresponding to the 5′ and 3′ sequences ofthe gene.

[0163] The 5′ primer for amplification of the hPrt1 coding sequence hasthe sequence: 5′ GACTTCTAGACCGCCATCATGCAGGACGCGGAGAACGTGGCG 3′ (SEQ IDNO:7) containing the underlined XbaI restriction enzyme site followed by24 bases of the sequence of the hPrt1 protein shown in FIGS. 1A-1D,beginning with the N-terminus of the protein. The 3′ primer has thesequence: 5′ GACTTCTAGAGGCGCAGGAGAAGGTGCCGCC 3′ (SEQ ID NO:8) containingthe underlined XbaI restriction site followed by 21 nucleotidescomplementary to the 3′ noncoding sequence shown in FIGS. 1A-1D.

[0164] The 5′ primer for amplification of the p97 coding sequence hasthe sequence: 5′ GACTGGTACCGCCATCATGGAGAGTGCGATTGCAGAAGGG 3′ (SEQ IDNO:9) containing the underlined Asp718 restriction enzyme site followedby 21 bases of the sequence of the p97 protein shown in FIGS. 2A-2E,beginning with the N-terminus of the protein. The 3′ primer has thesequence: 5′ GACTGGTACCCGCAGTGGTTAGGTCAAATGC 3′ (SEQ ID NO:10)containing the underlined Asp718 restriction site followed by 21nucleotides complementary to the 3′ noncoding sequence shown in FIGS.2A-2E.

[0165] The amplified fragment encoding either hPrt1 or p97 is isolatedfrom a 1% agarose gel using a commercially available kit (“Geneclean,”BIO 101 Inc., La Jolla, Calif.). The hPrt1 coding fragment then isdigested with XbaI and the p97 coding fragment then is digested withAsp718 . Each fragment is again is purified on a 1% agarose gel. Thesefragment are designated herein F1″.

[0166] The plasmid is digested with the restriction enzymes with eitherXbaI or Asp718 and optionally, can be dephosphorylated using calfintestinal phosphatase, using routine procedures known in the art. TheDNA is then isolated from a 1% agarose gel using a commerciallyavailable kit (“Geneclean” BIO 101 Inc., La Jolla, Calif.). This vectorDNA is designated herein “V1”.

[0167] Fragment F1 and the dephosphorylated plasmid V1 are ligatedtogether with T4 DNA ligase. E. coli HB101 or other suitable E. colihosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.)cells are transformed with the ligation mixture and spread on cultureplates. Bacteria are identified that contain the plasmid with either thehuman hPrt1 or p97 gene using the PCR method, in which one of theprimers that is used to amplify the gene and the second primer is fromwell within the vector so that only those bacterial colonies containingthe hPrt1 or p97 gene fragment will show amplification of the DNA. Thesequence of the cloned fragment is confirmed by DNA sequencing. Theseplasmids are designated herein pBac hPrt1 and pBac(p97).

[0168] Five μg of either pBac hPrt1 and pBac(p97) plasmid isco-transfected with 1.0 μg of a commercially available linearizedbaculovirus DNA (“BaculoGold baculovirus DNA”, Pharmingen, San Diego,Calif.), using the lipofection method described by Felgner et al., Proc.Natl. Acad. Sci. USA 84:7413-7417 (1987). 1 μg of BaculoGold virus DNAand 5 μg of the plasmid are mixed in a sterile well of a microtiterplate containing 50 μl of serum-free Grace's medium (Invitrogen LifeTechnologies, Carlsbad, Calif.). Afterwards, 10 μI Lipofectin plus 90 μlGrace's medium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is rocked back and forth tomix the newly added solution. The plate is then incubated for 5 hours at27° C. After 5 hours the transfection solution is removed from the plateand 1 ml of Grace's insect medium supplemented with 10% fetal calf serumis added. The plate is put back into an incubator and cultivation iscontinued at 27° C. for four days.

[0169] After four days the supernatant is collected and a plaque assayis performed, as described by Summers and Smith, supra. An agarose gelwith “Blue Gal” (Invitrogen Life Technologies, Carlsbad, Calif.) is usedto allow easy identification and isolation of gal-expressing clones,which produce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed bylnvitrogen LifeTechnologies, Carlsbad, Calif., page 9-10). After appropriateincubation, blue stained plaques are picked with the tip of amicropipettor (e.g., Eppendorf). The agar containing the recombinantviruses is then resuspended in a microcentrifuge tube containing 200 μlof Grace's medium and the suspension containing the recombinantbaculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Fourdays later the supernatants of these culture dishes are harvested andthen they are stored at 4° C. The recombinant viruses are called V-hPrt1and V-p97.

[0170] To verify the expression of the hPrt1 and p97 genes, Sf9 cellsare grown in Grace's medium supplemented with 10% heat inactivated FBS.The cells are infected with the recombinant baculovirus V-hPrt1 or V-p97at a multiplicity of infection (“MOI”) of about 2. Six hours later themedium is removed and is replaced with SF900 II medium minus methionineand cysteine (available from Invitrogen Life Technologies, Carlsbad,Calif.). If radiolabeled proteins are desired, 42 hours later, 5 μCi of³⁵S-methionine and 5 μCi ³⁵S-cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then they areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled). Microsequencing of the amino acidsequence of the amino terminus of purified protein may be used todetermine the amino terminal sequence of the mature protein and thus thecleavage point and length of the secretory signal peptide.

Example 4

[0171] Cloning and Expression of hPrt1 and p97 in Mammalian Cells

[0172] A typical mammalian expression vector contains the promoterelement, which mediates the initiation of transcription of mRNA, theprotein coding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as PSVL and PMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be usedinclude, human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

[0173] Alternatively, the gene can be expressed in stable cell linesthat contain the gene integrated into a chromosome. The co-transfectionwith a selectable marker such as dhfr, gpt, neomycin, or hygromycinallows the identification and isolation of the transfected cells.

[0174] The transfected gene can also be amplified to express largeamounts of the encoded protein. The DHFR (dihydrofolate reductase)marker is useful to develop cell lines that carry several hundred oreven several thousand copies of the gene of interest. Another usefulselection marker is the enzyme glutamine synthase (GS) (Murphy et al.,Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology10:169-175 (1992)). Using these markers, the mammalian cells are grownin selective medium and the cells with the highest resistance areselected. These cell lines contain the amplified gene(s) integrated intoa chromosome. Chinese hamster ovary (CHO) and NSO cells are often usedfor the production of proteins.

[0175] The expression vectors pC1 and pC4 contain the strong promoter(LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and CellularBiology, 438447 (March, 1985)) plus a fragment of the CMV-enhancer(Boshart et al., Cell 41:521-530 (1985)). Multiple cloning sites, e.g.,with the restriction enzyme cleavage sites BamHI, XbaI and Asp718,facilitate the cloning of the gene of interest. The vectors contain inaddition the 3′ intron, the polyadenylation and termination signal ofthe rat preproinsulin gene.

[0176] Example 4(a): Cloning and Expression in COS Cells

[0177] The expression plasmids, phPrt1 HA and p(p97) HA, are made bycloning cDNAs encoding the hPrt1 and p97 proteins into the expressionvector pcDNAI/Amp or pcDNAIII (which can be obtained from Invitrogen,Inc.).

[0178] The expression vector pcDNAII contains: (1) an E. coli origin ofreplication effective for propagation in E. coli and other prokaryoticcells; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) and a CMV promoter, apolylinker, an SV40 intron followed by a termination codon andpolyadenylation signal arranged so that a cDNA can be convenientlyplaced under expression control of the CMV promoter and operably linkedto the SV40 intron and the polyadenylation signal by means ofrestriction sites in the polylinker. The HA tag corresponds to anepitope derived from the influenza hemagglutinin protein described byWilson et al., Cell 37:767 (1984). The fusion of the HA tag to thetarget protein allows easy detection and recovery of the recombinantprotein with an antibody that recognizes the HA epitope. pcDNAIIIcontains, in addition, the selectable neomycin marker.

[0179] With respect to the hPrt1 protein, a DNA fragment encoding theprotein is cloned into the polylinker region of the vector so thatrecombinant protein expression is directed by the CMV promoter. Theplasmid construction strategy is as follows. The hPrt1 cDNA of thedeposited clone is amplified using primers that contain convenientrestriction sites, much as described above for construction of vectorsfor expression of hPrt1 protein in E. coli. Suitable primers include thefollowing, which are used in this example. The 5′ primer, containing theunderlined XbaI site, a Kozak sequence, an AUG start codon and 7additional codons of the 5′ coding region of the complete hPrt1 proteinhas the following sequence: 5 GACTTCTAGACCGCCATCATGCAGGACGCGGAGAACGTGGCG3 (SEQ ID NO:7). The 3′ primer, containing the underlined XbaI site, astop codon, the HA tag sequence, and 19 bp of 3′ coding sequence has thefollowing sequence (at the 3′ end): 5′GACTCTAGATTAAGCGTAGTCTGGGACGTCGTATGGGTAAA TCCCCCACTGCAGACAC 3′ (SEQ IDNO: 11).

[0180] Similarly, a DNA fragment encoding the p97 protein is cloned intothe polylinker region of the same vector. The plasmid constructionstrategy is as follows. The p97 cDNA of the deposited clone is alsoamplified using primers that contain convenient restriction sites.Suitable primers include the following, which are used in this example.The 5′ primer, containing the underlined Asp718 site, a Kozak sequence,an AUG start codon and 7 additional codons of the 5′ coding region ofthe complete p97 protein has the following sequence: 5′GACTGGTACCGCCATCATGGAGAGTGCGATTGCAGAAGGG 3′ (SEQ ID NO:9). The 3′primer, containing the underlined Asp718 site, a stop codon, the HA tagsequence, and 18 bp of 3′ coding sequence has the following sequence (atthe 3′ end): 5′ GACGGTACCTTAAGCGTAGTCTGGGACGTCGTATGGGTAGTCAGCTTCTTCCTCTGA 3′ (SEQ ID NO:12).

[0181] The PCR amplified DNA fragments and the vector, pcDNAIII, aredigested with XbaI for insertion of the hPrt1 coding sequences andAsp718 for insertion of the p97 coding sequences, and then ligated. Theligation mixture is transformed into E. Coli strain SURE (available fromStratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla,Calif. 92037), and the transformed culture is plated on ampicillin mediaplates which then are incubated to allow growth of ampicillin resistantcolonies. Plasmid DNA is isolated from resistant colonies and examinedby restriction analysis or other means for the presence of either thehPrt1 or p97 encoding fragment.

[0182] For expression of recombinant hPrt1 and p97, COS cells aretransfected with an expression vector, as described above, usingDEAE-DEXTRAN, as described, for instance, in Sambrook et al., MolecularCloning: a Laboratory Manual, Cold Spring Laboratory Press (1989). Cellsare incubated under conditions for expression of either hPrt1 or p97 bythe vector.

[0183] Expression of the hPrt1-HA or p97-HA fusion proteins is detectedby radiolabeling and immunoprecipitation, using methods described in,for example Harlow et al., Antibodies: A Laboratory Manual, 2nd Ed.;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York(1988). To this end, two days after transfection, the cells are labeledby incubation in media containing ³⁵S-cysteine for 8 hours. The cellsand the media are collected, and the cells are washed and lysed withdetergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5%DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. cited above.Proteins are precipitated from the cell lysate and from the culturemedia using an HA-specific monoclonal antibody. The precipitatedproteins then are analyzed by SDS-PAGE and autoradiography. Anexpression product of the expected size is seen in the cell lysate,which is not seen in negative controls.

[0184] Example 4(b): Cloning and Expression in CHO Cells

[0185] The vector pC4 is used for the expression of hPrt1 and p97proteins. Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCCAccession No. 37146). The plasmid contains the mouse DHFR gene undercontrol of the SV40 early promoter. Chinese hamster ovary- or othercells lacking dihydrofolate activity that are transfected with theseplasmids can be selected by growing the cells in a selective medium(alpha minus MEM, Invitrogen Life Technologies) supplemented with thechemotherapeutic agent methotrexate. The amplification of the DHFR genesin cells resistant to methotrexate (MTX) has been well documented (see,e.g., Alt, F. W., Kellems, R. M., Bertino, J. R., and Schimke, R. T.,1978, J Biol. Chem. 253:1357-1370, Hamlin, J. L. and Ma, C. 1990,Biochem. et Biophys. Acta, 1097:107-143, Page, M. J. and Sydenham, M. A.1991, Biotechnology 9:64-68). Cells grown in increasing concentrationsof MTX develop resistance to the drug by overproducing the targetenzyme, DHFR, as a result of amplification of the DHFR gene. If a secondgene is linked to the DHFR gene, it is usually co-amplified andover-expressed. It is known in the art that this approach may be used todevelop cell lines carrying more than 1,000 copies of the amplifiedgene(s). Subsequently, when the methotrexate is withdrawn, cell linesare obtained which contain the amplified gene integrated into one ormore chromosome(s) of the host cell.

[0186] Plasmid pC4 contains for expressing the gene of interest thestrong promoter of the long terminal repeat (LTR) of the Rous SarcomaVirus (Cullen, et al., Molecular and Cellular Biology, March1985:438-447) plus a fragment isolated from the enhancer of theimmediate early gene of human cytomegalovirus (CMV) (Boshart et al.,Cell 41:521-530 (1985)). Downstream of the promoter is a BamHIrestriction enzyme cleavage site that allows integration of the genes.Behind this cloning site the plasmid contains the 3′ intron andpolyadenylation site of the rat preproinsulin gene. Other highefficiency promoters can also be used for the expression, e.g., thehuman -actin promoter, the SV40 early or late promoters or the longterminal repeats from other retroviruses, e.g., HIV and HTLVI.Clontech's Tet-Off and Tet-On gene expression systems and similarsystems can be used to express the hPrt1 or p97 proteins in a regulatedway in mammalian cells (Gossen, M., & Bujard, H. 1992, Proc. Natl. Acad.Sci. USA 89: 5547-5551). For the polyadenylation of the MRNA othersignals, e.g., from the human growth hormone or globin genes can be usedas well. Stable cell lines carrying a gene of interest integrated intothe chromosomes can also be selected upon co-transfection with aselectable marker such as gpt, G418 or hygromycin. It is advantageous touse more than one selectable marker in the beginning, e.g., G418 plusmethotrexate.

[0187] The plasmid pC4 is digested with the restriction enzymes XbaI forinsertion of the hPrt1 encoding fragment and Asp718 for insertion of thep97 encoding fragment. The vectors are then dephosphorylated using calfintestinal phosphatase by procedures known in the art. The vectors arethen isolated from a 1% agarose gel.

[0188] The DNA sequence encoding the complete hPrt1 protein is amplifiedusing PCR oligonucleotide primers corresponding to the 5′ and 3′sequences of the gene. The 5′ primer has the sequence: 5GACTTCTAGACCGCCATCATGCAGGACGCGGAGAACGTGGCG 3 (SEQ ID NO:7) containingthe underlined XbaI restriction enzyme site followed by an efficientsignal for initiation of translation in eukaryotes, as described byKozak, M., J Mol. Biol. 196:947-950 (1987), and 24 bases of the codingsequence of hPrt1 cDNA shown in FIGS. 1A-1D (SEQ ID NO: 1). The 3′primer has the sequence: 5′ GACTTCTAGAGGCGCAGGAGAAGGTGCCGCC 3 (SEQ IDNO:8) containing the underlined XbaI restriction site followed by 21nucleotides complementary to the non-translated region of the hPrt1 geneshown in FIGS. 1A-1D (SEQ ID NO: 1).

[0189] Similarly, the DNA sequence encoding the complete p97 protein isalso amplified using PCR oligonucleotide primers corresponding to the 5′and 3′ sequences of the gene. The 5′ primer has the sequence: 5′GACTGGTACCGCCATCATGGAGAGTGCGATTGCAGAAGGG 3′ (SEQ ID NO:9) containing theunderlined Asp718 restriction enzyme site followed by an efficientsignal for initiation of translation in eukaryotes, as described byKozak, M., J. Mol. Biol. 196:947-950 (1987), and 24 bases of the codingsequence of p97 cDNA shown in FIGS. 2A-2E (SEQ ID NO:3). The 3′ primerhas the sequence: 5′ GACTGGTACCCGCAGTGGTTAGGTCAAATGC 3 (SEQ ID NO:10)containing the underlined Asp718 restriction site followed by 21nucleotides complementary to the non-translated region of the p97 geneshown in FIGS. 2A-2E (SEQ ID NO:3).

[0190] The amplified fragments are then digested with the endonucleasesXbaI for insertion of the hPrt1 encoding fragment and Asp718 forinsertion of the p97 encoding fragment and then purified again on a 1%agarose gel. The isolated fragments and the dephosphorylated vectors arethen ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells arethen transformed and bacteria are identified that contain the fragmentinserted into plasmid pC4 using, for instance, restriction enzymeanalysis.

[0191] Chinese hamster ovary cells lacking an active DHFR gene are usedfor transfection. 5 μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSV2-neo using lipofectin (Felgner et al.,supra). The plasmid pSV2neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reverse phase HPLCanalysis.

Example 5

[0192] Tissue distribution of hPrt1 and p97 mRNA expression

[0193] Northern blot analysis is carried out to examine hPrt1 and p97gene expression in human tissues, using methods described by, amongothers, Sambrook et al., cited above. A cDNA probe containing the entirenucleotide sequence of the hPrt1 (SEQ ID NO: 1) or p97 (SEQ ID NO: 3)protein is labeled with ³²p using the rediprime DNA labeling system(Amersham Life Science), according to manufacturer's instructions. Afterlabeling, the probe is purified using a CHROMA SPIN-100 column (ClontechLaboratories, Inc.), according to manufacturer's protocol numberPT1200-1. The purified labeled probe is then used to examine varioushuman tissues for either hPrt1 or p97 mRNA.

[0194] Multiple Tissue Northern (MTN) blots containing various humantissues (H) or human immune system tissues (IN) are obtained fromClontech and are examined with the labeled probe using ExpressHybhybridization solution (Clontech) according to manufacturer's protocolnumber PT1190-1. Following hybridization and washing, the blots aremounted and exposed to film at −70° C. overnight, and films developedaccording to standard procedures.

[0195] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples.

[0196] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the appended claims.

[0197] The entire disclosure of all publications (including patents,patent applications, journal articles, laboratory manuals, books, orother documents) cited herein are hereby incorporated by reference.

1 13 1 3032 DNA Homo sapiens CDS (97)..(2718) 1 ccctcgagtc gacggtatcgataagcttat cgataccgtc gactgctacc gaaggccggc 60 ggccgcggag ccctgcgagtaggcagcgtt gggccc atg cag gac gcg gag aac 114 Met Gln Asp Ala Glu Asn 15 gtg gcg gtg ccc gag gcg gcc gag gag cgc gcc gag ccc ggc cag cag 162Val Ala Val Pro Glu Ala Ala Glu Glu Arg Ala Glu Pro Gly Gln Gln 10 15 20cag ccg gcc gcc gag ccg ccg cca gcc gag ggg ctg ctg cgg ccc gcg 210 GlnPro Ala Ala Glu Pro Pro Pro Ala Glu Gly Leu Leu Arg Pro Ala 25 30 35 gggccc ggc gct ccg gag gcc gcg ggg acc gag gcc tcc agt gag gag 258 Gly ProGly Ala Pro Glu Ala Ala Gly Thr Glu Ala Ser Ser Glu Glu 40 45 50 gtg gggatc gcg gag gcc ggg ccg gag ccc gag gtg agg acc gag ccg 306 Val Gly IleAla Glu Ala Gly Pro Glu Pro Glu Val Arg Thr Glu Pro 55 60 65 70 gcg gccgag gca gag gcg gcc tcc ggc ccg tcc gag tcg ccc tcg ccg 354 Ala Ala GluAla Glu Ala Ala Ser Gly Pro Ser Glu Ser Pro Ser Pro 75 80 85 ccg gcc gccgag gag ctg ccc ggg tcg cat gct gag ccc cct gtc ccg 402 Pro Ala Ala GluGlu Leu Pro Gly Ser His Ala Glu Pro Pro Val Pro 90 95 100 gca cag ggcgag gcc cca gga gag cag gct cgg gac gca ggc tcc gac 450 Ala Gln Gly GluAla Pro Gly Glu Gln Ala Arg Asp Ala Gly Ser Asp 105 110 115 agc cgg gcccag gcg gtg tcc gag gac gcg gga gga aac gag ggc aga 498 Ser Arg Ala GlnAla Val Ser Glu Asp Ala Gly Gly Asn Glu Gly Arg 120 125 130 gcg gcc gaggcc gaa ccc cgg gcg ctg gag aac ggc gac gcg gac gag 546 Ala Ala Glu AlaGlu Pro Arg Ala Leu Glu Asn Gly Asp Ala Asp Glu 135 140 145 150 ccc tccttc agc gac ccc gag gac ttc gtg gac gac gtg agc gag gaa 594 Pro Ser PheSer Asp Pro Glu Asp Phe Val Asp Asp Val Ser Glu Glu 155 160 165 gaa ttactg gga gat gta ctc aaa gat cgg ccc cag gaa gca gat gga 642 Glu Leu LeuGly Asp Val Leu Lys Asp Arg Pro Gln Glu Ala Asp Gly 170 175 180 atc gattcg gtg att gta gtg gac aat gtc cct cag gtg gga ccc gac 690 Ile Asp SerVal Ile Val Val Asp Asn Val Pro Gln Val Gly Pro Asp 185 190 195 cga cttgag aaa ctc aaa aat gtc atc cac aag atc ttt tcc aag ttt 738 Arg Leu GluLys Leu Lys Asn Val Ile His Lys Ile Phe Ser Lys Phe 200 205 210 ggg aaaatc aca aat gat ttt tat cct gaa gag gat ggg aag aca aaa 786 Gly Lys IleThr Asn Asp Phe Tyr Pro Glu Glu Asp Gly Lys Thr Lys 215 220 225 230 gggtat att ttc ctg gag tac gcg tcc cct gcc cac gct gtg gat gct 834 Gly TyrIle Phe Leu Glu Tyr Ala Ser Pro Ala His Ala Val Asp Ala 235 240 245 gtgaag aac gcc gac ggc tac aag ctt gac aag cag cac aca ttc cgg 882 Val LysAsn Ala Asp Gly Tyr Lys Leu Asp Lys Gln His Thr Phe Arg 250 255 260 gtcaac ctc ttt acg gat ttt gac aag tat atg acg atc agt gac gag 930 Val AsnLeu Phe Thr Asp Phe Asp Lys Tyr Met Thr Ile Ser Asp Glu 265 270 275 tgggat att cca gag aaa cag cct ttc aaa gac ctg ggg aac tta cgt 978 Trp AspIle Pro Glu Lys Gln Pro Phe Lys Asp Leu Gly Asn Leu Arg 280 285 290 tactgg ctt gaa gag gca gaa tgc aga gat cag tac agt gtg att ttt 1026 Tyr TrpLeu Glu Glu Ala Glu Cys Arg Asp Gln Tyr Ser Val Ile Phe 295 300 305 310gag agt gga gac cgc act tcc ata ttc tgg aat gac gta aaa gac cct 1074 GluSer Gly Asp Arg Thr Ser Ile Phe Trp Asn Asp Val Lys Asp Pro 315 320 325gtc tca att gaa gaa aga gcg aga tgg aca gag acg tat gtg cgt tgg 1122 ValSer Ile Glu Glu Arg Ala Arg Trp Thr Glu Thr Tyr Val Arg Trp 330 335 340tct cct aag ggc acc tac ctg gct acc ttt cat caa aga ggc att gct 1170 SerPro Lys Gly Thr Tyr Leu Ala Thr Phe His Gln Arg Gly Ile Ala 345 350 355cta tgg ggg gga gag aaa ttc aag caa att cag aga ttc agc cac caa 1218 LeuTrp Gly Gly Glu Lys Phe Lys Gln Ile Gln Arg Phe Ser His Gln 360 365 370ggg gtt cag ctt att gac ttc tca cct tgt gaa agg tac ctg gtg acc 1266 GlyVal Gln Leu Ile Asp Phe Ser Pro Cys Glu Arg Tyr Leu Val Thr 375 380 385390 ttt agc ccc ctg atg gac acg cag gat gac cct cag gcc ata atc atc 1314Phe Ser Pro Leu Met Asp Thr Gln Asp Asp Pro Gln Ala Ile Ile Ile 395 400405 tgg gac atc ctt acg ggg cac aag aag agg ggt ttt cac tgt gag agc 1362Trp Asp Ile Leu Thr Gly His Lys Lys Arg Gly Phe His Cys Glu Ser 410 415420 tca gcc cat tgg cct att ttt aag tgg agc cat gat ggc aaa ttc ttt 1410Ser Ala His Trp Pro Ile Phe Lys Trp Ser His Asp Gly Lys Phe Phe 425 430435 gcc aga atg acc ctg gat acg ctt agc atc tat gaa act cct tct atg 1458Ala Arg Met Thr Leu Asp Thr Leu Ser Ile Tyr Glu Thr Pro Ser Met 440 445450 ggt ctt ttg gac aag aag agt ttg aag atc tct ggg ata aaa gac ttt 1506Gly Leu Leu Asp Lys Lys Ser Leu Lys Ile Ser Gly Ile Lys Asp Phe 455 460465 470 tct tgg tct cct ggt ggt aac ata atc gcc ttc tgg gtg cct gaa gac1554 Ser Trp Ser Pro Gly Gly Asn Ile Ile Ala Phe Trp Val Pro Glu Asp 475480 485 aaa gat att cca gcc agg gta acc ctg atg cag ctc cct acc agg caa1602 Lys Asp Ile Pro Ala Arg Val Thr Leu Met Gln Leu Pro Thr Arg Gln 490495 500 gag atc cga gtg agg aac ctg ttc aat gtg gtg gac tgc aag ctc cat1650 Glu Ile Arg Val Arg Asn Leu Phe Asn Val Val Asp Cys Lys Leu His 505510 515 tgg cag aag aac gga gac tac ttg tgt gtg aaa gta gat agg act ccg1698 Trp Gln Lys Asn Gly Asp Tyr Leu Cys Val Lys Val Asp Arg Thr Pro 520525 530 aaa ggc acc cag ggt gtt gtc aca aat ttt gaa att ttc cga atg agg1746 Lys Gly Thr Gln Gly Val Val Thr Asn Phe Glu Ile Phe Arg Met Arg 535540 545 550 gag aaa cag gta cct gtg gat gtg gtc gag atg aaa gaa acc atcata 1794 Glu Lys Gln Val Pro Val Asp Val Val Glu Met Lys Glu Thr Ile Ile555 560 565 gcc ttt gcc tgg gaa cca aat gga agt aag ttt gct gtg ctg cacgga 1842 Ala Phe Ala Trp Glu Pro Asn Gly Ser Lys Phe Ala Val Leu His Gly570 575 580 gag gct ccg cgg ata tct gtg tct ttc tac cac gtc aaa aac aacggg 1890 Glu Ala Pro Arg Ile Ser Val Ser Phe Tyr His Val Lys Asn Asn Gly585 590 595 aag att gaa ctc atc aag atg ttc gac aag cag cag gcg aac accatc 1938 Lys Ile Glu Leu Ile Lys Met Phe Asp Lys Gln Gln Ala Asn Thr Ile600 605 610 ttc tgg agc ccc caa gga cag ttc gtg gtg ttg gcg ggc ctg aggagt 1986 Phe Trp Ser Pro Gln Gly Gln Phe Val Val Leu Ala Gly Leu Arg Ser615 620 625 630 atg aac ggt gcc tta gcg ttt gtg gac act tcg gac tgc acggtc atg 2034 Met Asn Gly Ala Leu Ala Phe Val Asp Thr Ser Asp Cys Thr ValMet 635 640 645 aac atc gca gag cac tac atg gct tcc gac gtc gaa tgg gatcct act 2082 Asn Ile Ala Glu His Tyr Met Ala Ser Asp Val Glu Trp Asp ProThr 650 655 660 ggg cgc tac gtc gtc acc tct gtg tcc tgg tgg agc cat aaggtg gac 2130 Gly Arg Tyr Val Val Thr Ser Val Ser Trp Trp Ser His Lys ValAsp 665 670 675 aac gcg tac tgg ctg tgg act ttc cag gga cgc ctc ctg cagaag aac 2178 Asn Ala Tyr Trp Leu Trp Thr Phe Gln Gly Arg Leu Leu Gln LysAsn 680 685 690 aac aag gac cgc ttc tgc cag ctg ctg tgg cgg ccc cgg cctccc aca 2226 Asn Lys Asp Arg Phe Cys Gln Leu Leu Trp Arg Pro Arg Pro ProThr 695 700 705 710 ctc ctg agc cag gaa cag atc aag caa att aaa aag gatctg aag aaa 2274 Leu Leu Ser Gln Glu Gln Ile Lys Gln Ile Lys Lys Asp LeuLys Lys 715 720 725 tac tct aag atc ttt gaa cag aag gat cgt ttg agt cagtcc aaa gcc 2322 Tyr Ser Lys Ile Phe Glu Gln Lys Asp Arg Leu Ser Gln SerLys Ala 730 735 740 tca aag gaa ttg gtg gag aga agg cgc acc atg atg gaagat ttc cgg 2370 Ser Lys Glu Leu Val Glu Arg Arg Arg Thr Met Met Glu AspPhe Arg 745 750 755 aag tac cgg aaa atg gcc cag gag ctc tat atg gag cagaaa aac gag 2418 Lys Tyr Arg Lys Met Ala Gln Glu Leu Tyr Met Glu Gln LysAsn Glu 760 765 770 cgc ctg gag ttg cga gga ggg gtg gac act gac gag ctggac agc aac 2466 Arg Leu Glu Leu Arg Gly Gly Val Asp Thr Asp Glu Leu AspSer Asn 775 780 785 790 gtg gac gac tgg gaa gag gag acc att gag ttc ttcgtc act gaa gaa 2514 Val Asp Asp Trp Glu Glu Glu Thr Ile Glu Phe Phe ValThr Glu Glu 795 800 805 atc att ccc ctc gga atc agg agt gac ctg gag cactgt gcg cag ccg 2562 Ile Ile Pro Leu Gly Ile Arg Ser Asp Leu Glu His CysAla Gln Pro 810 815 820 tgt gtg ctg tgg agc cga ggc cgt cct gca gga agccgc gtg act ccc 2610 Cys Val Leu Trp Ser Arg Gly Arg Pro Ala Gly Ser ArgVal Thr Pro 825 830 835 gcc tcc tcc ctg tgc tct ctg gct ctg gac tgt gactgc gcc tgg att 2658 Ala Ser Ser Leu Cys Ser Leu Ala Leu Asp Cys Asp CysAla Trp Ile 840 845 850 ctg cca ttg cga cac att ttt gtg cct ttc agc ccctgg tgt ctg cag 2706 Leu Pro Leu Arg His Ile Phe Val Pro Phe Ser Pro TrpCys Leu Gln 855 860 865 870 tgg ggg att taa ggcacccgct tccacttctttcttgtttgg agttttctgt 2758 Trp Gly Ile tggaaccgcc ggcgttggct ccgaagacttagcgacgcac tggcggcacc ttctcctgcg 2818 cccagtgatg tttccacggt gcctgtacacagccgagcag catttccgtt gaaggacttg 2878 catccccatt gcgggcagtg ctggacgtgtcccggagacc caccggaggg cgccgcatgc 2938 cttgtacccc caccgtgcag gttgtggccggttttctccg caggttgaac atggaaataa 2998 aagcaaactt gtatgaaaaa aaaaaaaaaaaaaa 3032 2 873 PRT Homo sapiens 2 Met Gln Asp Ala Glu Asn Val Ala ValPro Glu Ala Ala Glu Glu Arg 1 5 10 15 Ala Glu Pro Gly Gln Gln Gln ProAla Ala Glu Pro Pro Pro Ala Glu 20 25 30 Gly Leu Leu Arg Pro Ala Gly ProGly Ala Pro Glu Ala Ala Gly Thr 35 40 45 Glu Ala Ser Ser Glu Glu Val GlyIle Ala Glu Ala Gly Pro Glu Pro 50 55 60 Glu Val Arg Thr Glu Pro Ala AlaGlu Ala Glu Ala Ala Ser Gly Pro 65 70 75 80 Ser Glu Ser Pro Ser Pro ProAla Ala Glu Glu Leu Pro Gly Ser His 85 90 95 Ala Glu Pro Pro Val Pro AlaGln Gly Glu Ala Pro Gly Glu Gln Ala 100 105 110 Arg Asp Ala Gly Ser AspSer Arg Ala Gln Ala Val Ser Glu Asp Ala 115 120 125 Gly Gly Asn Glu GlyArg Ala Ala Glu Ala Glu Pro Arg Ala Leu Glu 130 135 140 Asn Gly Asp AlaAsp Glu Pro Ser Phe Ser Asp Pro Glu Asp Phe Val 145 150 155 160 Asp AspVal Ser Glu Glu Glu Leu Leu Gly Asp Val Leu Lys Asp Arg 165 170 175 ProGln Glu Ala Asp Gly Ile Asp Ser Val Ile Val Val Asp Asn Val 180 185 190Pro Gln Val Gly Pro Asp Arg Leu Glu Lys Leu Lys Asn Val Ile His 195 200205 Lys Ile Phe Ser Lys Phe Gly Lys Ile Thr Asn Asp Phe Tyr Pro Glu 210215 220 Glu Asp Gly Lys Thr Lys Gly Tyr Ile Phe Leu Glu Tyr Ala Ser Pro225 230 235 240 Ala His Ala Val Asp Ala Val Lys Asn Ala Asp Gly Tyr LysLeu Asp 245 250 255 Lys Gln His Thr Phe Arg Val Asn Leu Phe Thr Asp PheAsp Lys Tyr 260 265 270 Met Thr Ile Ser Asp Glu Trp Asp Ile Pro Glu LysGln Pro Phe Lys 275 280 285 Asp Leu Gly Asn Leu Arg Tyr Trp Leu Glu GluAla Glu Cys Arg Asp 290 295 300 Gln Tyr Ser Val Ile Phe Glu Ser Gly AspArg Thr Ser Ile Phe Trp 305 310 315 320 Asn Asp Val Lys Asp Pro Val SerIle Glu Glu Arg Ala Arg Trp Thr 325 330 335 Glu Thr Tyr Val Arg Trp SerPro Lys Gly Thr Tyr Leu Ala Thr Phe 340 345 350 His Gln Arg Gly Ile AlaLeu Trp Gly Gly Glu Lys Phe Lys Gln Ile 355 360 365 Gln Arg Phe Ser HisGln Gly Val Gln Leu Ile Asp Phe Ser Pro Cys 370 375 380 Glu Arg Tyr LeuVal Thr Phe Ser Pro Leu Met Asp Thr Gln Asp Asp 385 390 395 400 Pro GlnAla Ile Ile Ile Trp Asp Ile Leu Thr Gly His Lys Lys Arg 405 410 415 GlyPhe His Cys Glu Ser Ser Ala His Trp Pro Ile Phe Lys Trp Ser 420 425 430His Asp Gly Lys Phe Phe Ala Arg Met Thr Leu Asp Thr Leu Ser Ile 435 440445 Tyr Glu Thr Pro Ser Met Gly Leu Leu Asp Lys Lys Ser Leu Lys Ile 450455 460 Ser Gly Ile Lys Asp Phe Ser Trp Ser Pro Gly Gly Asn Ile Ile Ala465 470 475 480 Phe Trp Val Pro Glu Asp Lys Asp Ile Pro Ala Arg Val ThrLeu Met 485 490 495 Gln Leu Pro Thr Arg Gln Glu Ile Arg Val Arg Asn LeuPhe Asn Val 500 505 510 Val Asp Cys Lys Leu His Trp Gln Lys Asn Gly AspTyr Leu Cys Val 515 520 525 Lys Val Asp Arg Thr Pro Lys Gly Thr Gln GlyVal Val Thr Asn Phe 530 535 540 Glu Ile Phe Arg Met Arg Glu Lys Gln ValPro Val Asp Val Val Glu 545 550 555 560 Met Lys Glu Thr Ile Ile Ala PheAla Trp Glu Pro Asn Gly Ser Lys 565 570 575 Phe Ala Val Leu His Gly GluAla Pro Arg Ile Ser Val Ser Phe Tyr 580 585 590 His Val Lys Asn Asn GlyLys Ile Glu Leu Ile Lys Met Phe Asp Lys 595 600 605 Gln Gln Ala Asn ThrIle Phe Trp Ser Pro Gln Gly Gln Phe Val Val 610 615 620 Leu Ala Gly LeuArg Ser Met Asn Gly Ala Leu Ala Phe Val Asp Thr 625 630 635 640 Ser AspCys Thr Val Met Asn Ile Ala Glu His Tyr Met Ala Ser Asp 645 650 655 ValGlu Trp Asp Pro Thr Gly Arg Tyr Val Val Thr Ser Val Ser Trp 660 665 670Trp Ser His Lys Val Asp Asn Ala Tyr Trp Leu Trp Thr Phe Gln Gly 675 680685 Arg Leu Leu Gln Lys Asn Asn Lys Asp Arg Phe Cys Gln Leu Leu Trp 690695 700 Arg Pro Arg Pro Pro Thr Leu Leu Ser Gln Glu Gln Ile Lys Gln Ile705 710 715 720 Lys Lys Asp Leu Lys Lys Tyr Ser Lys Ile Phe Glu Gln LysAsp Arg 725 730 735 Leu Ser Gln Ser Lys Ala Ser Lys Glu Leu Val Glu ArgArg Arg Thr 740 745 750 Met Met Glu Asp Phe Arg Lys Tyr Arg Lys Met AlaGln Glu Leu Tyr 755 760 765 Met Glu Gln Lys Asn Glu Arg Leu Glu Leu ArgGly Gly Val Asp Thr 770 775 780 Asp Glu Leu Asp Ser Asn Val Asp Asp TrpGlu Glu Glu Thr Ile Glu 785 790 795 800 Phe Phe Val Thr Glu Glu Ile IlePro Leu Gly Ile Arg Ser Asp Leu 805 810 815 Glu His Cys Ala Gln Pro CysVal Leu Trp Ser Arg Gly Arg Pro Ala 820 825 830 Gly Ser Arg Val Thr ProAla Ser Ser Leu Cys Ser Leu Ala Leu Asp 835 840 845 Cys Asp Cys Ala TrpIle Leu Pro Leu Arg His Ile Phe Val Pro Phe 850 855 860 Ser Pro Trp CysLeu Gln Trp Gly Ile 865 870 3 3820 DNA Homo sapiens CDS (307)..(3030) 3cagcagtgag tcggagctct atggaggtgg cagcgggtac cgagtggcgg ctgcagcagc 60gactcctctg agctgagttt gaggccgtcc ccgactcctt cctccccctt ccctccccct 120tttttttgtt ttccgttccc ctttcccctc ccttccctat ccccgacgac cggatcctga 180ggaggcagct gcggtggcag ctgctgagtt ctcggtgaag gtatttcatt tctcctgtcc 240cctcccctcc ccaccccatc tattaatatt attcttttga agattcttcg ttgtcaagcc 300gccaaa gtg gag agt gcg att gca gaa ggg ggt gct tct cgt ttc agt 348 ValGlu Ser Ala Ile Ala Glu Gly Gly Ala Ser Arg Phe Ser 1 5 10 gct tct tcgggc gga gga gga agt agg ggt gca cct cag cac tat ccc 396 Ala Ser Ser GlyGly Gly Gly Ser Arg Gly Ala Pro Gln His Tyr Pro 15 20 25 30 aag act gctggc aac agc gag ttc ctg ggg aaa acc cca ggg caa aac 444 Lys Thr Ala GlyAsn Ser Glu Phe Leu Gly Lys Thr Pro Gly Gln Asn 35 40 45 gct cag aaa tggatt cct gca cga agc act aga cga gat gac aac tcc 492 Ala Gln Lys Trp IlePro Ala Arg Ser Thr Arg Arg Asp Asp Asn Ser 50 55 60 gca gca aac aac tccgca aac gaa aaa gaa cga cat gat gca atc ttc 540 Ala Ala Asn Asn Ser AlaAsn Glu Lys Glu Arg His Asp Ala Ile Phe 65 70 75 agg aaa gta aga ggc atacta aat aag ctt act cct gaa aag ttt gac 588 Arg Lys Val Arg Gly Ile LeuAsn Lys Leu Thr Pro Glu Lys Phe Asp 80 85 90 aag cta tgc ctt gag ctc ctcaat gtg ggt gta gag tct aaa ctc atc 636 Lys Leu Cys Leu Glu Leu Leu AsnVal Gly Val Glu Ser Lys Leu Ile 95 100 105 110 ctt aaa ggg gtc ata ctgctg att gtg gac aaa gcc cta gaa gag cca 684 Leu Lys Gly Val Ile Leu LeuIle Val Asp Lys Ala Leu Glu Glu Pro 115 120 125 aag tat agc tca ctg tatgct cag cta tgt ctg cga ttg gca gaa gat 732 Lys Tyr Ser Ser Leu Tyr AlaGln Leu Cys Leu Arg Leu Ala Glu Asp 130 135 140 gca cca aac ttt gat ggccca gca gca gag ggt caa cca gga cag aag 780 Ala Pro Asn Phe Asp Gly ProAla Ala Glu Gly Gln Pro Gly Gln Lys 145 150 155 caa agc acc aca ttc agacgc ctc cta att tcc aaa tta caa gat gaa 828 Gln Ser Thr Thr Phe Arg ArgLeu Leu Ile Ser Lys Leu Gln Asp Glu 160 165 170 ttt gaa aac cga act agaaat gtt gat gtc tat gat aag cgt gaa aat 876 Phe Glu Asn Arg Thr Arg AsnVal Asp Val Tyr Asp Lys Arg Glu Asn 175 180 185 190 ccc ctc ctc ccc gaggag gag gaa cag aga gcc att gct aag atc aag 924 Pro Leu Leu Pro Glu GluGlu Glu Gln Arg Ala Ile Ala Lys Ile Lys 195 200 205 atg ttg gga aac atcaaa ttc att gga gag ctt ggc aag ctt gat ctt 972 Met Leu Gly Asn Ile LysPhe Ile Gly Glu Leu Gly Lys Leu Asp Leu 210 215 220 att cac gaa tct atcctt cat aag tgc atc aaa aca ctt ttg gaa aag 1020 Ile His Glu Ser Ile LeuHis Lys Cys Ile Lys Thr Leu Leu Glu Lys 225 230 235 aag aag aga gtc caactc aaa gat atg gga gag gat ttg gag tgc ctc 1068 Lys Lys Arg Val Gln LeuLys Asp Met Gly Glu Asp Leu Glu Cys Leu 240 245 250 tgt cag ata atg aggaca gtg gga cct aga tta gac cat gaa cga gcc 1116 Cys Gln Ile Met Arg ThrVal Gly Pro Arg Leu Asp His Glu Arg Ala 255 260 265 270 aag tcc tta atggat cag tac ttt gcc cga atg tgc tcc ttg atg tta 1164 Lys Ser Leu Met AspGln Tyr Phe Ala Arg Met Cys Ser Leu Met Leu 275 280 285 agt aag gaa ttgcca gca agg att cgt ttc ctg ctg cag gat acc gta 1212 Ser Lys Glu Leu ProAla Arg Ile Arg Phe Leu Leu Gln Asp Thr Val 290 295 300 gag ttg cga gaacac cat tgg gtt cct cgc aag gct ttt ctt gac aat 1260 Glu Leu Arg Glu HisHis Trp Val Pro Arg Lys Ala Phe Leu Asp Asn 305 310 315 gga cca aag acgatc aat caa att cgt caa gat gca gta aaa gat cta 1308 Gly Pro Lys Thr IleAsn Gln Ile Arg Gln Asp Ala Val Lys Asp Leu 320 325 330 ggg gtg ttt attcct gct cct atg gct caa ggg atg aga agt gac ttc 1356 Gly Val Phe Ile ProAla Pro Met Ala Gln Gly Met Arg Ser Asp Phe 335 340 345 350 ttt ctg gaggga ccg ttc atg cca ccc agg atg aaa atg gat agg gac 1404 Phe Leu Glu GlyPro Phe Met Pro Pro Arg Met Lys Met Asp Arg Asp 355 360 365 cca ctt ggagga ctt gct gat atg ttt gga caa atg cca ggt agc gga 1452 Pro Leu Gly GlyLeu Ala Asp Met Phe Gly Gln Met Pro Gly Ser Gly 370 375 380 att ggt actggt cca gga gtt atc cag gat aga ttt tca ccc acc atg 1500 Ile Gly Thr GlyPro Gly Val Ile Gln Asp Arg Phe Ser Pro Thr Met 385 390 395 gga cgt catcgt tca aat caa ctc ttc aat ggc cat ggg gga cac atc 1548 Gly Arg His ArgSer Asn Gln Leu Phe Asn Gly His Gly Gly His Ile 400 405 410 atg cct cccaca caa tcg cag ttt gga gag atg gga ggc aag ttt atg 1596 Met Pro Pro ThrGln Ser Gln Phe Gly Glu Met Gly Gly Lys Phe Met 415 420 425 430 aaa agccag ggg cta agc cag ctc tac cat aac cag agt cag gga ctc 1644 Lys Ser GlnGly Leu Ser Gln Leu Tyr His Asn Gln Ser Gln Gly Leu 435 440 445 tta tcccag ctg caa gga cag tcg aag gat atg cca cct cgg ttt tct 1692 Leu Ser GlnLeu Gln Gly Gln Ser Lys Asp Met Pro Pro Arg Phe Ser 450 455 460 aag aaagga cag ctt aat gca gat gag att agc ctg agg cct gct cag 1740 Lys Lys GlyGln Leu Asn Ala Asp Glu Ile Ser Leu Arg Pro Ala Gln 465 470 475 tcg ttccta atg aat aaa aat caa gtg cca aag ctt cag ccc cag ata 1788 Ser Phe LeuMet Asn Lys Asn Gln Val Pro Lys Leu Gln Pro Gln Ile 480 485 490 act atgatt cct cct agt gca caa cca cca cgc act caa aca cca cct 1836 Thr Met IlePro Pro Ser Ala Gln Pro Pro Arg Thr Gln Thr Pro Pro 495 500 505 510 ctggga cag aca cct cag ctt ggt ctc aaa act aat cca cca ctt atc 1884 Leu GlyGln Thr Pro Gln Leu Gly Leu Lys Thr Asn Pro Pro Leu Ile 515 520 525 caggaa aag cct gcc aag acc agc aaa aag cca cca ccg tca aag gaa 1932 Gln GluLys Pro Ala Lys Thr Ser Lys Lys Pro Pro Pro Ser Lys Glu 530 535 540 gaactc ctt aaa cta act gaa act gtt gtg act gaa tat cta aat agt 1980 Glu LeuLeu Lys Leu Thr Glu Thr Val Val Thr Glu Tyr Leu Asn Ser 545 550 555 ggaaat gca aat gag gct gtc aat ggt gta aga gaa atg agg gct cct 2028 Gly AsnAla Asn Glu Ala Val Asn Gly Val Arg Glu Met Arg Ala Pro 560 565 570 aaacac ttt ctt cct gag atg tta agc aaa gta atc atc ctg tca cta 2076 Lys HisPhe Leu Pro Glu Met Leu Ser Lys Val Ile Ile Leu Ser Leu 575 580 585 590gat aga agc gat gaa gat aaa gaa aaa gca agt tct ttg atc agt tta 2124 AspArg Ser Asp Glu Asp Lys Glu Lys Ala Ser Ser Leu Ile Ser Leu 595 600 605ctc aaa cag gaa ggg ata gcc aca agt gac aac ttc atg cag gct ttc 2172 LeuLys Gln Glu Gly Ile Ala Thr Ser Asp Asn Phe Met Gln Ala Phe 610 615 620ctg aat gta ttg gac cag tgt ccc aaa ctg gag gtt gac atc cct ttg 2220 LeuAsn Val Leu Asp Gln Cys Pro Lys Leu Glu Val Asp Ile Pro Leu 625 630 635gtg aaa tcc tat tta gca cag ttt gca gct cgt gcc atc att tca gag 2268 ValLys Ser Tyr Leu Ala Gln Phe Ala Ala Arg Ala Ile Ile Ser Glu 640 645 650ctg gtg agc att tca gaa cta gct caa cca cta gaa agt ggc acc cat 2316 LeuVal Ser Ile Ser Glu Leu Ala Gln Pro Leu Glu Ser Gly Thr His 655 660 665670 ttt cct ctc ttc cta ctt tgt ctt cag cag tta gct aaa tta caa gat 2364Phe Pro Leu Phe Leu Leu Cys Leu Gln Gln Leu Ala Lys Leu Gln Asp 675 680685 cga gaa tgg tta aca gaa ctt ttt caa caa agc aag gtc aat atg cag 2412Arg Glu Trp Leu Thr Glu Leu Phe Gln Gln Ser Lys Val Asn Met Gln 690 695700 aaa atg ctc cca gaa att gat cag aat aag gac cgc atg ttg gag att 2460Lys Met Leu Pro Glu Ile Asp Gln Asn Lys Asp Arg Met Leu Glu Ile 705 710715 ttg gaa gga aag gga ctg agt ttc tta ttc cca ctc ctc aaa ttg gag 2508Leu Glu Gly Lys Gly Leu Ser Phe Leu Phe Pro Leu Leu Lys Leu Glu 720 725730 aag gaa ctg ttg aag caa ata aag ttg gat cca tcc cct caa acc ata 2556Lys Glu Leu Leu Lys Gln Ile Lys Leu Asp Pro Ser Pro Gln Thr Ile 735 740745 750 tat aaa tgg att aaa gat aac atc tct ccc aaa ctt cat gta gat aaa2604 Tyr Lys Trp Ile Lys Asp Asn Ile Ser Pro Lys Leu His Val Asp Lys 755760 765 gga ttt gtg aac atc tta atg act agc ttc tta cag tac att tct agt2652 Gly Phe Val Asn Ile Leu Met Thr Ser Phe Leu Gln Tyr Ile Ser Ser 770775 780 gaa gta aac ccc ccc agc gat gaa aca gat tca tcc tct gct cct tcc2700 Glu Val Asn Pro Pro Ser Asp Glu Thr Asp Ser Ser Ser Ala Pro Ser 785790 795 aaa gaa cag tta gag cag gaa aaa caa cta cta cta tct ttc aag cca2748 Lys Glu Gln Leu Glu Gln Glu Lys Gln Leu Leu Leu Ser Phe Lys Pro 800805 810 gta atg cag aaa ttt ctt cat gat cac gtt gat cta caa gtc agt gcc2796 Val Met Gln Lys Phe Leu His Asp His Val Asp Leu Gln Val Ser Ala 815820 825 830 ctg tat gct ctc cag gtg cac tgc tat aac agc aac ttc cca aaaggc 2844 Leu Tyr Ala Leu Gln Val His Cys Tyr Asn Ser Asn Phe Pro Lys Gly835 840 845 atg tta ctt cgc ttt ttt gtg cac ttc tat gac atg gaa att attgaa 2892 Met Leu Leu Arg Phe Phe Val His Phe Tyr Asp Met Glu Ile Ile Glu850 855 860 gaa gaa gct ttc ttg gct tgg aaa gaa gat ata acc caa gag tttccg 2940 Glu Glu Ala Phe Leu Ala Trp Lys Glu Asp Ile Thr Gln Glu Phe Pro865 870 875 gga aaa ggc aag gct ttg ttc cag gtg aat cag tgg cta acc tggtta 2988 Gly Lys Gly Lys Ala Leu Phe Gln Val Asn Gln Trp Leu Thr Trp Leu880 885 890 gaa act gct gaa gaa gaa gaa tca gag gaa gaa gct gac taa 3030Glu Thr Ala Glu Glu Glu Glu Ser Glu Glu Glu Ala Asp 895 900 905agaaccagcc aaagccttaa attgtgcaaa acatactgtt gctatgatgt aactgcattt 3090gacctaacca ctgcgaaaat tcattccgct gtaatgtttt cacaatattt aaagcagaag 3150cacgtcagtt aggatttcct tctgcataag gtttttttgt agtgtaatgt cttaatcata 3210gtctaccatc aaatatttta ggagtatctt taatgtttag atagtatatt agcagcatgc 3270aataattaca tcataagttc tcaagcagag gcagtctatt gcaaggacct tctttgctgc 3330cagttatcat aggctgtttt aagttagaaa actgaatagc aacactgaat actgtagaaa 3390tgcactttgc tcagtaatac ttgagttgtt gcaatatttg attatccatt tggttgttac 3450agaaaaattc ttaactgtaa ttgatggttg ttgccgtaat agtatattgc ctgtatttct 3510acctctagta atgggcttta tgtgctagat tttaatatcc ttgagcctgg gcaagtgcac 3570aagtcttttt aaaagaaaca tggtttactt gcacaaaact gatcagtttt gagagatcgt 3630taatgccctt gaagtggttt ttgtgggtgt gaaacaaatg gtgagaattt gaattggtcc 3690ctcctattat agtattgaaa ttaagtctac ttaatttatc aagtcatgtt catgccctga 3750ttttatatac ttgtatctat caataaacat tgtgatactt gaaaaaaaaa aaaaaaaaaa 3810aaaaaaaaaa 3820 4 907 PRT Homo sapiens 4 Val Glu Ser Ala Ile Ala Glu GlyGly Ala Ser Arg Phe Ser Ala Ser 1 5 10 15 Ser Gly Gly Gly Gly Ser ArgGly Ala Pro Gln His Tyr Pro Lys Thr 20 25 30 Ala Gly Asn Ser Glu Phe LeuGly Lys Thr Pro Gly Gln Asn Ala Gln 35 40 45 Lys Trp Ile Pro Ala Arg SerThr Arg Arg Asp Asp Asn Ser Ala Ala 50 55 60 Asn Asn Ser Ala Asn Glu LysGlu Arg His Asp Ala Ile Phe Arg Lys 65 70 75 80 Val Arg Gly Ile Leu AsnLys Leu Thr Pro Glu Lys Phe Asp Lys Leu 85 90 95 Cys Leu Glu Leu Leu AsnVal Gly Val Glu Ser Lys Leu Ile Leu Lys 100 105 110 Gly Val Ile Leu LeuIle Val Asp Lys Ala Leu Glu Glu Pro Lys Tyr 115 120 125 Ser Ser Leu TyrAla Gln Leu Cys Leu Arg Leu Ala Glu Asp Ala Pro 130 135 140 Asn Phe AspGly Pro Ala Ala Glu Gly Gln Pro Gly Gln Lys Gln Ser 145 150 155 160 ThrThr Phe Arg Arg Leu Leu Ile Ser Lys Leu Gln Asp Glu Phe Glu 165 170 175Asn Arg Thr Arg Asn Val Asp Val Tyr Asp Lys Arg Glu Asn Pro Leu 180 185190 Leu Pro Glu Glu Glu Glu Gln Arg Ala Ile Ala Lys Ile Lys Met Leu 195200 205 Gly Asn Ile Lys Phe Ile Gly Glu Leu Gly Lys Leu Asp Leu Ile His210 215 220 Glu Ser Ile Leu His Lys Cys Ile Lys Thr Leu Leu Glu Lys LysLys 225 230 235 240 Arg Val Gln Leu Lys Asp Met Gly Glu Asp Leu Glu CysLeu Cys Gln 245 250 255 Ile Met Arg Thr Val Gly Pro Arg Leu Asp His GluArg Ala Lys Ser 260 265 270 Leu Met Asp Gln Tyr Phe Ala Arg Met Cys SerLeu Met Leu Ser Lys 275 280 285 Glu Leu Pro Ala Arg Ile Arg Phe Leu LeuGln Asp Thr Val Glu Leu 290 295 300 Arg Glu His His Trp Val Pro Arg LysAla Phe Leu Asp Asn Gly Pro 305 310 315 320 Lys Thr Ile Asn Gln Ile ArgGln Asp Ala Val Lys Asp Leu Gly Val 325 330 335 Phe Ile Pro Ala Pro MetAla Gln Gly Met Arg Ser Asp Phe Phe Leu 340 345 350 Glu Gly Pro Phe MetPro Pro Arg Met Lys Met Asp Arg Asp Pro Leu 355 360 365 Gly Gly Leu AlaAsp Met Phe Gly Gln Met Pro Gly Ser Gly Ile Gly 370 375 380 Thr Gly ProGly Val Ile Gln Asp Arg Phe Ser Pro Thr Met Gly Arg 385 390 395 400 HisArg Ser Asn Gln Leu Phe Asn Gly His Gly Gly His Ile Met Pro 405 410 415Pro Thr Gln Ser Gln Phe Gly Glu Met Gly Gly Lys Phe Met Lys Ser 420 425430 Gln Gly Leu Ser Gln Leu Tyr His Asn Gln Ser Gln Gly Leu Leu Ser 435440 445 Gln Leu Gln Gly Gln Ser Lys Asp Met Pro Pro Arg Phe Ser Lys Lys450 455 460 Gly Gln Leu Asn Ala Asp Glu Ile Ser Leu Arg Pro Ala Gln SerPhe 465 470 475 480 Leu Met Asn Lys Asn Gln Val Pro Lys Leu Gln Pro GlnIle Thr Met 485 490 495 Ile Pro Pro Ser Ala Gln Pro Pro Arg Thr Gln ThrPro Pro Leu Gly 500 505 510 Gln Thr Pro Gln Leu Gly Leu Lys Thr Asn ProPro Leu Ile Gln Glu 515 520 525 Lys Pro Ala Lys Thr Ser Lys Lys Pro ProPro Ser Lys Glu Glu Leu 530 535 540 Leu Lys Leu Thr Glu Thr Val Val ThrGlu Tyr Leu Asn Ser Gly Asn 545 550 555 560 Ala Asn Glu Ala Val Asn GlyVal Arg Glu Met Arg Ala Pro Lys His 565 570 575 Phe Leu Pro Glu Met LeuSer Lys Val Ile Ile Leu Ser Leu Asp Arg 580 585 590 Ser Asp Glu Asp LysGlu Lys Ala Ser Ser Leu Ile Ser Leu Leu Lys 595 600 605 Gln Glu Gly IleAla Thr Ser Asp Asn Phe Met Gln Ala Phe Leu Asn 610 615 620 Val Leu AspGln Cys Pro Lys Leu Glu Val Asp Ile Pro Leu Val Lys 625 630 635 640 SerTyr Leu Ala Gln Phe Ala Ala Arg Ala Ile Ile Ser Glu Leu Val 645 650 655Ser Ile Ser Glu Leu Ala Gln Pro Leu Glu Ser Gly Thr His Phe Pro 660 665670 Leu Phe Leu Leu Cys Leu Gln Gln Leu Ala Lys Leu Gln Asp Arg Glu 675680 685 Trp Leu Thr Glu Leu Phe Gln Gln Ser Lys Val Asn Met Gln Lys Met690 695 700 Leu Pro Glu Ile Asp Gln Asn Lys Asp Arg Met Leu Glu Ile LeuGlu 705 710 715 720 Gly Lys Gly Leu Ser Phe Leu Phe Pro Leu Leu Lys LeuGlu Lys Glu 725 730 735 Leu Leu Lys Gln Ile Lys Leu Asp Pro Ser Pro GlnThr Ile Tyr Lys 740 745 750 Trp Ile Lys Asp Asn Ile Ser Pro Lys Leu HisVal Asp Lys Gly Phe 755 760 765 Val Asn Ile Leu Met Thr Ser Phe Leu GlnTyr Ile Ser Ser Glu Val 770 775 780 Asn Pro Pro Ser Asp Glu Thr Asp SerSer Ser Ala Pro Ser Lys Glu 785 790 795 800 Gln Leu Glu Gln Glu Lys GlnLeu Leu Leu Ser Phe Lys Pro Val Met 805 810 815 Gln Lys Phe Leu His AspHis Val Asp Leu Gln Val Ser Ala Leu Tyr 820 825 830 Ala Leu Gln Val HisCys Tyr Asn Ser Asn Phe Pro Lys Gly Met Leu 835 840 845 Leu Arg Phe PheVal His Phe Tyr Asp Met Glu Ile Ile Glu Glu Glu 850 855 860 Ala Phe LeuAla Trp Lys Glu Asp Ile Thr Gln Glu Phe Pro Gly Lys 865 870 875 880 GlyLys Ala Leu Phe Gln Val Asn Gln Trp Leu Thr Trp Leu Glu Thr 885 890 895Ala Glu Glu Glu Glu Ser Glu Glu Glu Ala Asp 900 905 5 33 DNA ArtificialSequence Forward 5′ primer for pAR90 N90146 5 accggaattc aaaatggacgcggacgagcc ctc 33 6 28 DNA Artificial Sequence Reverse 3′ primer forpAR90 N90146 6 agcggaattc ttaaatcccc cactgcag 28 7 42 DNA ArtificialSequence Contains XbaI restriction enzyme site 7 gacttctaga ccgccatcatgcaggacgcg gagaacgtgg cg 42 8 31 DNA Artificial Sequence Contains XbaIrestriction enzyme site 8 gacttctaga ggcgcaggag aaggtgccgc c 31 9 40 DNAArtificial Sequence Contains Asp718 restriction enzyme site 9 gactggtaccgccatcatgg agagtgcgat tgcagaaggg 40 10 31 DNA Artificial SequenceContains Asp718 restriction enzyme site 10 gactggtacc cgcagtggttaggtcaaatg c 31 11 58 DNA Artificial Sequence Contains an XbaI site, astop codon, and an HA tag sequence 11 gactctagat taagcgtagt ctgggacgtcgtatgggtaa atcccccact gcagacac 58 12 57 DNA Artificial Sequence Containsan Asp718 site, a stop codon, and an HA tag sequence 12 gacggtaccttaagcgtagt ctgggacgtc gtatgggtag tcagcttctt cctctga 57 13 10 PRT Homosapiens 13 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Gly 1 5 10

What is claimed is:
 1. An isolated nucleic acid molecule comprising apolynucleotide having a nucleotide sequence at least 95% identical to asequence selected from the group consisting of: (a) a nucleotidesequence encoding the hPrt1 polypeptide having the complete amino acidsequence in SEQ ID NO:2; (b) a nucleotide sequence encoding the hPrt1polypeptide having the amino acid sequence at positions from about 2 toabout 873 in SEQ ID NO:2; (c) a nucleotide sequence encoding the hPrt1polypeptide having the complete amino acid sequence encoded by the cDNAclone contained in ATCC Deposit Number 97766; (d) a nucleotide sequenceencoding the p97 polypeptide having the complete amino acid sequence inSEQ ID NO:4; (e) a nucleotide sequence encoding the p97 polypeptidehaving the amino acid sequence at positions from about 2 to about 907 inSEQ ID NO:4; (f) a nucleotide sequence encoding the p97 polypeptidehaving the complete amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit Number 97767; and (g) a nucleotide sequencecomplementary to any of the nucleotide sequences in (a), (b), (c), (d),(e) or (f).
 2. The nucleic acid molecule of claim 1 wherein saidpolynucleotide has the complete nucleotide sequence in SEQ ID NO: 1 orSEQ ID NO:3.
 3. The nucleic acid molecule of claim 1 wherein saidpolynucleotide has either the nucleotide sequence in SEQ ID NO: 1encoding a polypeptide having the complete amino acid sequence in SEQ IDNO:2 or the nucleotide sequence in SEQ ID NO:3 encoding a polypeptidehaving the complete amino acid sequence in SEQ ID NO:4.
 4. The nucleicacid molecule of claim 1 wherein said polynucleotide has the completenucleotide sequence of the cDNA clones contained in ATCC Deposit Number97766 or ATCC Deposit Number
 97767. 5. The nucleic acid molecule ofclaim 1 wherein said polynucleotide has the nucleotide sequence encodingthe polypeptides having the complete amino acid sequence encoded by theCDNA clones contained in ATCC Deposit Number 97766 or ATCC DepositNumber
 97767. 6. An isolated nucleic acid molecule comprising apolynucleotide which hybridizes under stringent hybridization conditionsto a polynucleotide having a nucleotide sequence identical to anucleotide sequence in (a), (b), (c), (d), (e), (f) or (g) of claim 1wherein said polynucleotide which hybridizes does not hybridize understringent hybridization conditions to a polynucleotide having anucleotide sequence consisting of only A residues or of only T residues.7. An isolated nucleic acid molecule comprising a polynucleotide whichencodes the amino acid sequence of an epitope-bearing portion of apolypeptide having an amino acid sequence in (a), (b), (c), (d), (e) or(f) of claim
 1. 8. The isolated nucleic acid molecule of claim 7, whichencodes an epitope-bearing portion of an hPrt1 polypeptide selected fromthe group consisting of: a polypeptide comprising amino acid residuesfrom about 1 to about 188 in SEQ ID NO:2; a polypeptide comprising aminoacid residues from about 193 to about 235 in SEQ ID NO:2; a polypeptidecomprising amino acid residues from about 248 to about 262 in SEQ IDNO:2; a polypeptide comprising amino acid residues from about 270 toabout 350 in SEQ ID NO:2; a polypeptide comprising amino acid residuesfrom about 361 to about 449 in SEQ ID NO:2; a polypeptide comprisingamino acid residues from about 458 to about 620 in SEQ ID NO:2; and apolypeptide comprising amino acid residues from about 639 to about 846in SEQ ID NO:2 or an epitope-bearing portion of a p97 polypeptideselected from the group consisting of: a polypeptide comprising aminoacid residues from about 1 to about 98 in SEQ ID NO:4; a polypeptidecomprising amino acid residues from about 121 to about 207 in SEQ IDNO:4; a polypeptide comprising amino acid residues from about 232 toabout 278 in SEQ ID NO:4; a polypeptide comprising amino acid residuesfrom about 287 to about 338 in SEQ ID NO:4; a polypeptide comprisingamino acid residues from about 347 to about 578 in SEQ ID NO:4; apolypeptide comprising amino acid residues from about 593 to about 639in SEQ ID NO:4; a polypeptide comprising amino acid residues from about681 to about 770 in SEQ ID NO:4; a polypeptide comprising amino acidresidues from about 782 to about 810 in SEQ ID NO:4; and a polypeptidecomprising amino acid residues from about 873 to about 905 in SEQ IDNO:4.
 9. A method for making a recombinant vector comprising insertingan isolated nucleic acid molecule of claim 1 into a vector.
 10. Arecombinant vector produced by the method of claim
 9. 11. A method ofmaking a recombinant host cell comprising introducing the recombinantvector of claim 10 into a host cell.
 12. A recombinant host cellproduced by the method of claim
 11. 13. A recombinant method forproducing a polypeptide, comprising culturing the recombinant host cellof claim 12 under conditions such that said polypeptide is expressed andrecovering said polypeptide.
 14. An isolated polypeptide having an aminoacid sequence at least 95% identical to a sequence selected from thegroup consisting of: (a) the amino acid sequence of the hPrt1polypeptide having the complete amino acid sequence in SEQ ID NO:2; (b)the amino acid sequence of the hPrt1 polypeptide having the amino acidsequence at positions from about 2 to about 873 in SEQ If) NO:2; (c) theamino acid sequence of the hPrt1 polypeptide having the complete aminoacid sequence encoded by the cDNA clone contained in ATCC Deposit Number97766; (d) the amino acid sequence of the p97 polypeptide having thecomplete amino acid sequence in SEQ ID NO:4; (e) the amino acid sequenceof the p97 polypeptide having the amino acid sequence at positions fromabout 2 to about 907 in SEQ ID NO:4; (f) the amino acid sequence of thep97 polypeptide having the complete amino acid sequence encoded by thecDNA clone contained in ATCC Deposit Number 97767; and (g) the aminoacid sequence of an epitope-bearing portion of any one of thepolypeptides of (a), (b), (c), (d), (e) or (f).
 15. An isolatedpolypeptide comprising an epitope-bearing portion of either the hPrt1protein, wherein said portion is selected from the group consisting of:a polypeptide comprising amino acid residues from about 1 to about 188in SEQ ID NO:2; a polypeptide comprising amino acid residues from about193 to about 235 in SEQ ID NO:2; a polypeptide comprising amino acidresidues from about 248 to about 262 in SEQ ID NO:2; a polypeptidecomprising amino acid residues from about 270 to about 350 in SEQ IDNO:2; a polypeptide comprising amino acid residues from about 361 toabout 449 in SEQ ID NO:2; a polypeptide comprising amino acid residuesfrom about 458 to about 620 in SEQ ID NO:2; and a polypeptide comprisingamino acid residues from about 639 to about 846 in SEQ ID NO:2 or thep97 protein, wherein said portion is selected from the group consistingof: a polypeptide comprising amino acid residues from about 1 to about98 in SEQ ID NO:4; a polypeptide comprising amino acid residues fromabout 121 to about 207 in SEQ ID NO:4; a polypeptide comprising aminoacid residues from about 232 to about 278 in SEQ ID NO:4; a polypeptidecomprising amino acid residues from about 287 to about 338 in SEQ IDNO:4; a polypeptide comprising amino acid residues from about 347 toabout 578 in SEQ ID NO:4; a polypeptide comprising amino acid residuesfrom about 593 to about 639 in SEQ ID NO:4; a polypeptide comprisingamino acid residues from about 681 to about 770 in SEQ ID NO:4; apolypeptide comprising amino acid residues from about 782 to about 810in SEQ ID NO:4; and a polypeptide comprising amino acid residues fromabout 873 to about 905 in SEQ ID NO:4.
 16. An isolated antibody thatbinds specifically to a polypeptide of claim
 15. 17. A method oftranslating mRNA in vitro, comprising: (a) a translation reactionmixture containing both labeled and unlabeled amino acids; (b) mixing aneffective amount of hPrt1 and/or p97 protein; (c) fractionating thesample to separate labeled protein; and (d) detecting the presence ofthe labeled protein.
 18. A method of screening for agonists andantagonists of hPrt1 and/or p97 activity comprising: (a) contactingcells which express hPrt1 and/or p97 polypeptides with a candidatecompound, (b) assaying a cellular response, and (c) comparing thecellular response to a standard cellular response made in absence of thecandidate compound; whereby, an increased cellular response over thestandard indicates that the compound is an agonist and a decreasedcellular response over the standard indicates that the compound is anantagonist.
 19. A method of treating a disease state associated withapoptosis comprising introducing an effective amount of an hPrt1 and/orp97 protein into an individual to be treated in admixture with apharmaceutically acceptable carrier.